Regional and TBX5-Dependent Gene Expression in the Atria: Implications for Pulmonary Vein Development and Atrial Fibrillation
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Atrial fibrillation, characterized by irregular atrial depolarization leading to an uncoordinated contraction of the atrial muscle, is the most common cardiac arrhythmia. The T-box transcription factor TBX5 regulates atrial rhythm and has been genetically associated with human atrial fibrillation by both familial and genome-wide association studies. Over the last 30 years, the pulmonary veins and peri-pulmonary region of the left atrium have been implicated as a physical locus that frequently initiates atrial fibrillation. Regional gene expression differences within the atria have been reported that may reflect distinct ontogeny of these regions and impact arrhythmia mechanisms. In this study, we investigated transcriptional differences between regions of the adult left atrium in the mouse. We performed and compared transcriptional profiling of the left atrial appendage and peri-pulmonary vein atrium of the adult mouse. We identified novel markers and transcriptional differences between the two regions, including of multiple known AF genes. Furthermore, we compared Tbx5-dependent genes between each region and identified Hcn4 as a novel dysregulated gene in the peri-pulmonary vein atrium. Regional and Tbx5-dependent gene expression differences between the left atrial appendage and peri-pulmonary vein atrium are considered in the context of the genetic basis of atrial fibrillation.
KeywordsAtrial fibrillation Arrhythmia TBX5 Cardiac conduction Pulmonary vein Atrium Gene regulatory network PITX2 HCN4 SHOX2
51.1.1 Atrial Fibrillation and the Pulmonary Vein
Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, affecting more than 7 million Americans and 33 million people worldwide . AF is characterized by an irregular pattern of atrial depolarization, resulting in rapid and disorganized atrial conduction and lack of effective atrial chamber contraction. The rhythm abnormality in AF manifests with circulatory deficits and systemic thromboembolism that greatly increase morbidity and mortality. Patients with AF have an increased risk of developing major complications including heart failure and stroke. In addition, the prevalence of AF is expected to rise significantly as the population ages. AF has become a major clinical and economic burden, owing to the limitations and side effects associated with current AF therapies. Although AF most often manifests in the context of pre-existing cardiac pathologies, idiopathic or lone AF forms indicated a heritable component . Over the last decade, genome-wide association studies (GWAS) have identified over one hundred AF-associated loci [3, 4]. These advances in understanding the genetic basis of AF portend an understanding of the complex molecular pathways provoking AF.
The current paradigm of AF requires two arrhythmogenic components: (1) ectopic (triggered) atrial myocardial activity such as early or delayed afterdepolarizations and (2) a fibrillogenic substrate that propagates these triggers throughout the atrial chambers . The most common source of triggered activity is the pulmonary veins, which connect the lungs to the left atria [6, 7]. It has been shown that the pulmonary vein (PV) and peri-pulmonary vein (PPV) myocardium can demonstrate automaticity which can trigger atrial depolarization . A pacemaker current has been observed in the PV-left atrial junction in atrial fibrillation, and gene expression contributing to myocardial automaticity, such as the HCN4 channel protein, is observed in the pulmonary veins . A common treatment for AF is to isolate the PV myocytes from the atrial myocardium by catheter ablation, severing the electrical connectivity between these two populations and preventing inappropriate PV depolarizations from initiating global atrial depolarization and atrial arrhythmias [6, 7].
An important advance in the understanding of the pathophysiology of AF has been the demonstration that the pulmonary vein plays an important role in the initiation of AF [6, 10]. Other initiating foci include the superior vena cava and the left atrium; however, the pulmonary vein remains the most common source of focal activity. This observation has stimulated consideration of the distinctions between the myocardium of the pulmonary/peri-pulmonary vein region and other regions of the atrium. However, the gene expression profile promoting focal activity around the PVs in humans has been difficult to elucidate, owing to the limitations of tissue samples from patients. The molecular identity of the PV myocardium, including specific ion channels and transcription factors expressed in this tissue, has only recently been described. Analysis of a whole-lung transcriptome data set uncovered a set of 24 transcripts, including sarcomeric structural proteins, genes regulating sarcomere assembly, ion transport proteins and hormone signaling in the pulmonary cardiomyocyte gene network . Some of these genes have been linked to AF in humans, suggesting that perturbation of this transcriptional network might lead to altered calcium handling, altered myocardium contractile force and electrophysiological properties which may contribute to the initiation of atrial arrhythmias.
51.1.2 TBX5 and Pulmonary Vein Development
Holt-Oram syndrome is an autosomal dominant disorder caused by mutations in the T-box family transcription factor TBX5 [12, 13, 14], occurring in approximately 1 in 100,000 live births [15, 16]. The clinical diagnosis of Holt-Oram syndrome includes completely penetrant upper-limb malformations and variable congenital heart defects, most commonly septal and cardiac conduction defects . Specifically, the conduction defects can manifest as long PR interval, AV and bundle branch block, bradycardia, sick sinus syndrome and atrial fibrillation with or without overt structural defects [18, 19]. The observation that conduction abnormalities can occur in the absence of structural heart defects was an early indication that TBX5 may play a direct role in controlling gene expression essential for normal cardiac conduction. Recently, GWAS have implicated TBX5 in altered cardiac conduction speed (PR and QRS intervals) and AF in structurally normal hearts. Although strict clinical diagnosis of Holt-Oram Syndrome does not involve defects of the pulmonary veins [14, 17], pulmonary vein abnormalities are often associated with Holt-Oram syndrome and TBX5 mutations [20, 21, 22]. Furthermore, homozygous mutation of Tbx5 in the mouse germline  results in complete morphological failure of the cardiopulmonary progenitors, which would normally generate portions of the atria, the atrial septum, pulmonary vein myocardium and smooth muscle cells and airways smooth muscle cells [24, 25]. These observations suggested that TBX5 may drive gene expression important for the development of the pulmonary vasculature in the embryo and for the suppression of atrial arrhythmias in the pulmonary veins or peri-pulmonary vein atrial myocardium in the adult.
Here, we describe regional differences between the transcriptome of the left atrial appendage and the peri-pulmonary vein of the left atrium from adult mice. RNA sequencing revealed previously known and novel regional differences, including several genes implicated in atrial fibrillation. We further compared Tbx5-depedent transcripts between the left atrial appendage and the peri-pulmonary vein of the left atrium from adult mice. Important distinctions were observed between the two regions including Hcn4, which demonstrated differential Tbx5-dependence. Our data suggests that underlying regional differences in the transcriptome of the atrial myocardium may enlighten susceptibility to atrial fibrillation.
51.2.1 Comparison of Peri-Pulmonary Vein and Atrial Appendage Transcriptomes
Because of the relationship between the left atria, pulmonary vein and AF, we examined region-specific gene expression against a list of 235 AF associated genes [3, 28, 29, 30]. Of the 606 genes that demonstrated regionalized expression in our RNA-seq, 19 have been associated with AF (Fig. 51.1d). Specifically, Adrb1, Nkx2-5, Tmem182, Nppa, Nppb, Gja5 and Kcnh7 are more strongly expressed in the LAA while Ntrk2, Tubb4a, Cacna1d, Tbx3, Scn3b, Shox2, Kcne4, Hcn4, Adra1a, Adrb3, Tubb3 and Cacna2d2 have higher expression in the PPV.
51.2.2 Tbx5-Dependent Transcriptome of Peri-Pulmonary Vein Atrium Identifies Genes Involved with Cardiac Conduction
We identified 338 significantly dysregulated genes (|log2FC| > 1; FDR < 0.01) comparing the PPV between Tbx5 conditional mutant and controls (Fig. 51.2b, GEO accession GSE128870). Of these, 149 were up-regulated and 189 were down-regulated in the Tbx5 mutants (Fig. 51.2b). Utilizing metascape , we identified seven representative GO terms associated with the up-regulated genes and two representative GO terms associated with the down-regulated genes (Fig. 51.2c). The down-regulated GO terms are reminiscent of the LAA previously reported . In particular, examining the full set of GO terms under “multicellular organismal signaling,” we identified multiple GO terms associated with the regulation of cardiac conduction, including ion channels, action potential and transmembrane signaling (Fig. 51.2d).
Genes displaying distinct Tbx5-dependent expression patterns
Skeletal muscle alpha-actin associated with myopathies 
Spindle assembly checkpoint and centromeric cohesion 
Mitotic kinesin protein involved in centromsome duplication 
Kinetochore-binding mitotic checkpoint gene 
Ventricular and slow skeletal muscle myosin light chain 
Inhibits inappropriate homologous recombination in mitosis 
Secretoglobin protein associated with anti-inflammatory function 
Removes lipid-derived aldehydes generated by oxidative stress 
Calcium-dependent, phospholipid-binding proteins 
Uncharacterized cardiac expressed, SH3 domain binding kinase 
Lung-expressed predicted gene 
Depolymerizes hyaluronic acid 
Type IX collagen found with type II fibrillar collagen 
Leukocyte associated integrin beta subunit 
Mitochondrial leucyl-tRNA synthetase 
Predicted gene 
Transmembrane protein with homology to Slit3 
Peroxidizes polyunsaturated fatty acids 
Membrane junction protein associated with axons 
Hyperpolarization-activated current/“funny” current 
Small Cajal body-specific RNA 10 ( )
Neuronal calcium sensor 
Antisense lncRNA 
Kidney-expressed antisense lncRNA 
Fetal growth hormone signaling and tissue differentiation 
Calcium-triggered vesicle transport 
Fatty acid transport 
Competing endogenous RNA to miR-182-5p 
51.3.1 The Genetics of Atrial Fibrillation Implicates Developmental Transcription Factors
Most genetic loci implicated in AF encode ion channels that affect arrhythmia trigger or substrate. Initial transcriptional studies of AF have focused on a restricted set of candidates, associating remodeling with changes in ion channels and components of cellular signaling cascades [31, 32]. Early transcriptomic or proteomic investigation of AF has been limited to animal models and/or chronic AF [33, 34, 35, 36]. For example, animal models based on disruption of specific ion channels do not exhibit spontaneous AF in the absence of concomitant cardiac pathophysiology . However, a recent transcriptomic study using the left atrial appendage of patients with AF revealed that AF susceptibility was associated with decreased expression of transcription factors involved in inflammation, oxidation and the cellular stress response . This study further suggested that remodeling of ion channel expression occurs later in the onset of the disease. Accumulating evidence suggests that transcription factors are important contributors to the pathogenesis of AF .
GWAS of AF have identified numerous transcription factor loci that play fundamental roles during cardiac development, including TBX5, NKX2-5, GATA4 and PITX2. The implication of transcription factor genes essential for cardiac development in in AF raises the fundamental question of whether AF is a function or a result of a developmental defect, for example, of the pulmonary veins, or a later manifestation of altered transcriptional control of cardiac rhythm genes in the adult. The first GWAS of AF identified a susceptibility locus on chromosome 4q25 adjacent to PITX2 . There is evidence that Pitx2c affects adult gene expression in the atrium. PITX2 is expressed both in the left atrium and the pulmonary myocardial sleeve and that its levels are decreased in patients with AF [41, 42]. Atrial-specific deletion of Pitx2c leads to hallmarks of AF, which is also observed in Pitx2c heterozygous mice or adult-specific deletion of this gene [41, 43, 44]. Furthermore, microarray analysis of Pitx2c heterozygous mice revealed impaired gap and tight junction gene expression, consistent with the hypothesis that structural genes are remodeled later in the onset of AF .
We previously demonstrated that adult-specific deletion of Tbx5 causes spontaneous and sustained AF with atrial gene regulatory network dysfunction . TBX5 drives the atrial expression of Pitx2, and TBX5 and PITX2 co-modulate the expression of cardiac rhythm effector genes, including Ryr2 and Atp2a2. These findings indicated that interactions between TBX5 and PITX2 provide tight control of an atrial rhythm gene regulatory network and that perturbation of this network triggered AF susceptibility. This example suggested that cardiac TFs implicated in AF by genetic association may co-regulate a gene regulatory network for atrial rhythm homeostasis in the adult atrium.
51.3.2 Transcription Factors and AF: A Pulmonary Vein Developmental Relationship?
The requirement of developmental TFs in the adult for normal cardiac rhythm control does not rule out the possibility that developmental defects caused by their deficiency in the embryo also contributes to AF susceptibility. During embryonic development, the cardiopulmonary progenitors give rise to parts of the atria, pulmonary vein and lungs . Distinct functional characteristics between the PPV and LAA regions may reflect distinct developmental ontogeny. The myocardium of the pulmonary veins has a distinct developmental origin from that of the atrial free walls . Subsequently, the myocardium surrounding the pulmonary veins is distinct from that of other regions in the atrium . These developmental distinctions may set the stage for regional gene expression differences within the mature atrium of the adult that normally maintains atrial rhythm but which make the PPV region susceptible to trigger formation when faced with environmental or genetic insult.
Mutations in NKX2-5, PITX2C and TBX5 increase susceptibility to AF, and all of these transcription factors (TF) play critical roles during cardiovascular development [26, 47, 48, 49, 50]. Each of these TFs are expressed in the pulmonary veins, suggesting that perturbations of the transcription program in the PV myocardium may underlie the pathogenesis of AF. The caudal myocardium, including the developing pulmonary vein myocardium, expresses Tbx5, Pitx2c, Nkx2-5 and Gja5 (Cx40) at high levels [51, 52] consistent with a fast-conducting atrial myocardial phenotype. In a Nkx2-5 haploinsufficient mouse model, Gja5 levels are decreased whereas Hcn4 levels are increased in the pulmonary vein myocardium. Nkx2-5 and Pitx2c are both required for the development and maintain identity of the pulmonary myocardium [45, 53]. The changes in gene expression observed in Nkx2-5 haploinsufficient mice could be reflective of a loss of fast conduction and acquisition of pacemaking conduction phenotype. This type of transition provided a conceivable model for how genetic changes could result in automaticity in the PPV region that resulted in arrhythmia initiation.
Absence of the lungs and pulmonary vasculature in Tbx5 knockout mice suggests that it may play an essential developmental role specifically within the pulmonary veins as well . We observed that a decrement in Tbx5 in the PPV region decreased Hcn4 expression, which is not overtly consistent with this transformation model. On the other hand, we observed that many genes with important roles in myocardial automaticity, including Tbx3, Shox2, Cacna1d, Cacna2d2 and Hcn4 were more highly expressed in the PPV than the LAA region, and remained more highly expressed in the context of adult-specific Tbx5 conditional deletion. Furthermore, other Tbx5-dependent genes in the PPV region are classical fast conduction genes, including Scn5a, Gja5 and Kcnk3. Reduction in the expression of these fast conduction genes in the context of retained high expression of automaticity genes such as Hcn4 could diminish fast conduction physiology and allow the emergence of myocardial automaticity, providing a nidus for the initiation of atrial arrhythmias including AF.
51.4.1 Animal Experiments and Ethics Statement
All murine experiments were performed under University of Chicago Institutional Animal Care and Use Committee (IACUC) protocol number 71737. Mice harboring the ROSA26 tamoxifen-inducible cre recombinase transgene, Gt(ROSA)26Sortm1(cre/ERT2)Tyj (R26creERT2), were bred with mice harboring the Tbx5tm1Jse allele (Tbx5flox) to generate Tbx5 conditional mutants, R26creERT2/creERT2; Tbx5flox/flox, and controls, R26creERT2/creERT2; Tbx5+/+ [23, 54]. Tamoxifen was administered by intraperitoneal injection for three consecutive days at 6 weeks of age and tissue samples were harvested 1 week after treatment as previously reported .
51.4.2 Transcriptional Profiling and Analysis
Transcriptional profiling was performed on the peri-pulmonary vein region of the left atrium (Fig. 51.1a). Total RNA was extracted from four controls and six conditional mutants. Following rRNA removal (Ribo-Zero rRNA Removal Kit, Illumina), libraries were prepared using the TruSeq RNA Sample prep kit v2 (Illumina). Samples were pooled in equimolar ratios and sequenced on the Illumina HiSeq4000 platform. Library preparation and sequencing were performed by the Genomics Core Facility at the University of Chicago.
The peri-pulmonary vein samples were compared with the previously published corresponding left atrial appendage samples . Both the peri-pulmonary vein and left atrial appendage samples were analyzed in parallel as described below.
Between 20 and 22 million reads were generated for each replicate (14–19 million reads for left atrial appendage) and aligned to the GRCm38/mm10 build of the Mus musculus genome (retrieved from UCSC May 23, 2012) using TopHat v2.1.1 [55, 56]. Reads were filtered to remove poorly aligned (MAPQ<10), duplicated and unmapped reads using BamTools v2.4.1 . Post-alignment, post-filtered reads were assigned to transcripts using StringTie v1.3.3 [58, 59].
Downstream analysis was performed using R v3.4.0 . Differential expression testing was performed using edgeR v3.18.1  and limma v3.32.10 . Low level genes were removed within each condition using median log-transformed counts per gene per million mapped reads (cpm) cutoff of 1. A generalized linear model (GLM) framework was used to test for differential expressed genes between Tbx5fl/fl;R26cre-ERT2 conditional mutants and Tbx5+/+;R26cre-ERT2 controls for each tissue and between controls of the left atrial appendage and peri-pulmonary vein tissues. Data was plotted using the ggplot2 v2.3.0  and scales v0.5.0  packages. GO term analysis was performed using metascape (http://metascape.org) . The raw and processed data as well as the full outputs were deposited in GEO at accession number GSE128870.
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