Date of Award

January 2023

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Vasanth Vedantham

Abstract

The sinoatrial node (SAN) consists of specialized pacemaker cardiomyocytes (PCs) that collectively function as the leading pacemaker of the heart. PCs have a distinct gene expression program that allows for automatic electrical activity and, importantly, coupling to the autonomic nervous system. Although the transcriptome of SAN pacemaker cells (PCs) has been defined, and it is well established that SAN function and arrhythmia susceptibility are heritable traits, the mechanisms whereby variation in non-coding regulatory elements influences cardiac conduction and arrhythmia susceptibility remain poorly studied. In particular, although it is well established that bradycardia is associated with the development of atrial fibrillation (AF), it is not known whether common genetic mechanisms lead to both AF and sinus node dysfunction.

In this study, we identify a non-coding regulatory element that affects both sinus node function and atrial fibrillation susceptibility. We first used Assay for Transposase Accessible Chromatin (ATAC-seq) in mouse pacemaker cells (PCs) and right-atrial cardiomyocytes (RACMs) to identify differentially accessible regions within the SAN. We then cross-referenced these SAN-specific regions with known common variants strongly associated with RHR in human populations. One such variant, rs17180489, (effect = -0.57 bpm per allele, p = 2.9e-74) was identified at a predicted GATA binding site within a conserved SAN enhancer located in an intron of the gene RGS6. The minor allele of this variant (MAF 0.15) abolishes the predicted GATA site and is negatively associated with HR response to exercise (effect = -0.4, p = 2.6e-06) and increases the risk for atrial fibrillation (OR (95% CI) = 1.03 (1.02-1.04), p = 9.6e-04). Because RGS6 modulates the response of cardiomyocytes to parasympathetic input, these results suggest a model whereby the minor allele results in reduced RGS6 expression in the SAN, due to loss of a GATA binding site, thereby regulating interaction of the SAN with the parasympathetic nervous system. We also hypothesized that the enhancer might have activity in the pulmonary vein myocardial sleeves (PVMS), thereby affecting triggered activity and refractoriness of the PVMS (known to trigger AF).

To test this model, we performed in-vivo and ex-vivo studies in a mouse line lacking the Rgs6 enhancer. Rgs6enh/enh mice were generated using CRISPR-Cas9-guided deletion of the 1.7-kb Rgs6 enhancer from the mouse genome. Rgs6enh/enh mice demonstrated a 61% reduction in Rgs6 mRNA levels within SAN tissue (p < 0.005) as assessed by qPCR, with no change in expression of any of the other 10 genes within the same topologically associated domain as Rgs6. Furthermore, our preliminary studies using hybridization chain reaction fluorescent in-situ hybridization (HCR RNA-FISH) on myocardial sections from Rgs6enh/enh adult mice and WT littermates revealed a qualitative decrease in RGS6 expression specifically within SAN pacemaker cells, with studies ongoing to assess expression in the PVMS and atria. Finally, in-vivo electrocardiography studies demonstrated that knockout mice had an exaggerated bradycardic response to carbachol (a muscarinic agonist) across both males (HR = -20.38 ± 1.37 bpm, p < 0.0001) and females (HR = -133.0 ± 1.33 bpm, p < 0.0001). These studies confirm that rs17180489 lies within a conserved Rgs6 enhancer that directly influences Rgs6 expression levels in the SAN and regulates the parasympathetic heart rate response, providing strong support for our model.

In addition to completing our experiments studying PVMS physiology, Rgs6 expression, and atrial fibrillation inducibility in Rgs6enh/enh mice, additional studies are underway to translate these findings to a human context. We are currently testing for enhancer activity differences between the major and minor alleles of rs17180489 iPSC-derived human pacemaker cardiomyocytes. Taken together, we anticipate that our findings will determine how a common variant affects the heritability of SAN function by modulating its interaction with the parasympathetic nervous system. We will test whether SAN function and atrial fibrillation susceptibility share a common genetic architecture due to overlapping molecular regulatory mechanisms in PCs and PVMS, a result that will have important implications for developing novel drug targets for sinus node dysfunction and atrial fibrillation, the most commonly encountered clinical arrhythmias.

Comments

This thesis is restricted to Yale network users only. This thesis is permanently embargoed from public release.

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