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. 2020 Dec;12(12):a037408. doi: 10.1101/cshperspect.a037408

Development of the Cardiac Conduction System

Samadrita Bhattacharyya 1, Nikhil V Munshi 1,2,3,4
PMCID: PMC7706568  PMID: 31988140

Abstract

The cardiac conduction system initiates and propagates each heartbeat. Specialized conducting cells are a well-conserved phenomenon across vertebrate evolution, although mammalian and avian species harbor specific components unique to organisms with four-chamber hearts. Early histological studies in mammals provided evidence for a dominant pacemaker within the right atrium and clarified the existence of the specialized muscular axis responsible for atrioventricular conduction. Building on these seminal observations, contemporary genetic techniques in a multitude of model organisms has characterized the developmental ontogeny, gene regulatory networks, and functional importance of individual anatomical compartments within the cardiac conduction system. This review describes in detail the transcriptional and regulatory networks that act during cardiac conduction system development and homeostasis with a particular emphasis on networks implicated in human electrical variation by large genome-wide association studies. We conclude with a discussion of the clinical implications of these studies and describe some future directions.

The primary function of the heart is to deliver blood to the systemic and pulmonary circuits. To execute this important function, the heart must integrate the physiology of a pump, an electrical wiring system, a series of barriers, and a set of one-way valves. Therefore, the mammalian heart consists of numerous individual cell types, including cardiomyocytes (CMs), fibroblasts, endothelial cells, immune cells, and others. CMs can be functionally categorized into contractile and conducting cells. Contractile cells constitute the vast majority (∼98%) of CMs in the heart and are primarily responsible for generating the force necessary for pumping blood throughout the body. In contrast, conducting cells comprise a rare subpopulation (∼2%) of CMs that constitute the cardiac conduction system (CCS), which generate and propagate the electrical impulse required to orchestrate cardiac contraction. In nearly all model organisms that have been studied, the first spontaneous action potentials are observed early during cardiac development (Kamino et al. 1981).

CCS ANATOMY

CCS components can be functionally classified into slow-conducting structures, including the sinoatrial node (SAN) and atrioventricular node (AVN), and the fast-conducting ventricular conduction system (VCS), which includes the atrioventricular bundle (AVB, also known as the His-Bundle), the right and left bundle branches (BBs), and the Purkinje fiber network (PFN) (Figs. 1 and 2). Pacemaker (PM) cells are highly specialized CMs with the intrinsic ability to rhythmically depolarize and initiate a cardiac impulse responsible for basal heart rate (HR). The SAN acts as the PM to initiate cardiac rhythm and depolarize the surrounding atrial CMs, leading to atrial systole. Before the onset of atrial systole, the impulse funnels toward the AVN, which delays the electric impulse to allow atrial contraction and late ventricular filling prior to ventricular systole to ensure optimal cardiac performance. After exiting the AVN, the impulse is rapidly propagated through the AVB and asymmetric BBs into the terminal PFN to directly depolarize working ventricular CMs resulting in ventricular contraction and expulsion of blood through the right and left ventricular outflow tracts. Any deviation from the normal sequence of events can cause clinically important arrhythmias.

Figure 1.

Figure 1.

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Functional anatomy of the cardiac conduction system (CCS). Red dotted lines indicate the direction of the electric impulse from sinoatrial node (SAN), to atrioventricular node (AVN), and then propagation through the ventricular conduction system (VCS), leading to ventricular contraction. Unique action potential tracings for each CCS compartment are shown in blue.

Figure 2.

Figure 2.

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Cellular and molecular networks for sinoatrial node (SAN) development. Upward pointing red arrows indicate high expression of genes. Yellow arrows indicate developmental stages of SAN specification, although additional contributions to the SAN are likely. Black arrows indicate activation of genes. Blue lines with blunt ends indicate suppression of gene expression by respective transcription factors (TFs). The blue dotted box represents a gene program that shuts down and the brown dotted box indicates a gene program that is activated during SAN maturation. The large continuous brown box indicates the gene regulatory networks at play during SAN development.

ELECTROCARDIOGRAM

Electrical impulse propagation through the heart is routinely visualized with an electrocardiogram (ECG or EKG). The P wave indicates atrial depolarization, the PR interval (or PQ interval) reflects atrioventricular (AV) depolarization and conduction, the QRS interval (from the beginning of the Q wave to the end of the S wave) reflects ventricular depolarization, and the T wave shows repolarization of the ventricles. Distinct electrocardiographic P wave and QRS complexes can be identified in the electrical activity of the embryonic avian and mammalian heart soon after the onset of chamber differentiation (van Mierop 1967). The RR interval is the time between QRS complexes. The instantaneous HR can be calculated from the time between two consecutive QRS complexes and reflects SAN activity. ECG parameters for normal electrical activity within each CCS compartment is provided in Figure 5 below.

Figure 5.

Figure 5.

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The anatomical location of each cardiac conduction system (CCS) compartment is shown with its corresponding electrophysiological activity on an electrocardiogram (ECG). The (HR)/RR interval for sinoatrial node (SAN) activity, PR parameters for atrial/atrioventricular node (AVN) activity, and QRS/QT parameters for ventricular conduction system (VCS) activity. Loci associated with various ECG traits are outlined by distinct colors: orange, SAN; light green, atrioventricular conduction system (AVCS) rhythm and disease; dark green, VCS activity or disease. Many of the genomic loci are associated with one or more of the above traits. (AVB) atrioventricular bundle, (BBs) bundle branches, (PFN) Purkinje fiber network.

GAP JUNCTIONS

In the mammalian heart, gap junction (GJ) channels mediate electric and metabolic communication between CMs, thereby allowing cardiac impulse propagation and coordinated contraction of the chambers. The spread of excitation in different parts of the heart is determined by tissue geometry, cellular excitability, and gap junctional coupling. The number, size, and distribution of GJ channel clusters, as well as the expression pattern of connexin isoforms, determine the electrophysiological properties of assembled GJ channels and cell-to-cell coupling.

NERVOUS SYSTEM CONTROL

In the absence of extrinsic neural or hormonal influences, the SAN is responsible for maintaining regular sinus rhythm between 60 and 100 beats per minute. During the “fight-or-flight” response, stimulation by the sympathetic nervous system activates β1 adrenergic receptors (expressed in the SAN, AVN, and on atrial and ventricular CMs), which increases HR (via the SAN), cardiac contractility, and AVN conduction velocity. The parasympathetic muscarinic M2 receptors are abundant in nodal and atrial tissue, but sparse in the ventricles. The binding of acetylcholine to M2 receptors normalizes sinus rhythm by slowing the rate of depolarization in the SAN and conduction velocity through the AVN. Additionally, activation of M2 receptors lowers atrial contractility, thus contributing to a reduction in stroke volume. Together, the effects of the parasympathetic nervous system on HR and stroke volume lead to an overall decrease in cardiac output (Gordan et al. 2015).

THE SAN

Anatomy and Primary Function

The SAN, which contains the primary cardiac PM, is located at the junction of the superior vena cava and the crista terminalis of the right atrium (RA). PM cells possess the unique capacity to trigger an action potential without external stimulation as compared with neighboring atrial contractile CMs.

Developmental Origin

The SAN anlage develops within the sinus venosus (SV), directly adjacent to the embryonic RA wall, and can be identified morphologically at ∼E10 in mouse (Virágh and Challice 1980). The definitive SV is comprised of the SAN, the sinus horns (left, common, and right), and the venous valves. However, the future dominant PM SAN cells have not yet been incorporated into the heart tube by E10 (Mommersteeg et al. 2010; Bressan et al. 2013). As the heart tube elongates by addition of posterior second heart field (SHF) progenitors, dominant PM activity moves toward the caudal pole, with a simultaneous caudal shift of Hcn4 (encoding potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4) expression (Garcia-Frigola et al. 2003). Findings from a seminal study suggest that chick PM cells arise from a unique mesodermal region outside of first heart field (FHF) and SHF, which requires further investigation in mammals. This discrete region is physically segregated and molecularly programmed in a “tertiary heart field” before cardiac morphogenesis (Burkhard et al. 2017).

Hcn4 is required for PM activity (Stieber et al. 2003). Interestingly, Hcn4 is also expressed in the early heart tube and serves as a dynamic marker of FHF (Liang et al. 2013). PM activity shifts into the SV on its formation, and from there to the anatomical SAN, during early fetal stages. In mice, CMs and their progenitors express Nkx2-5 until E9.0–E9.5, at which point the future atrial compartment has been established (Stanley et al. 2002). However, from E10 (definitive SAN anlage) until E12, the recruited progenitor cells express Tbx18 (encoding the T-box TF 18) instead of Nkx2-5 and develop into the Nkx2-5lowTbx18+ myocardium, which includes the SV and the definitive sinus node head (Christoffels et al. 2006). Similarly, Hcn4 expression is down-regulated in CMs that will form the atria, and becomes confined to the Nkx2-5lowTbx18+ SV and eventually the SAN (Mommersteeg et al. 2007a; Wiese et al. 2009). The gene for atrial natriuretic peptide, Nppa, is expressed in atrial myocytes but not in the SAN or other nodal cells, providing a complementary negative marker (Christoffels et al. 2010). Although TBX18 is not required for regulation of the SV/SAN gene program (Wiese et al. 2009), it promotes differentiation of sinus node myocardium in the dorsal pericardial wall.

Maintenance of Normal Electrical Activity in SAN

In the SAN, cell–cell coupling is mediated by Cx30.2 and Cx45 (encoded by the Gjd3 and Gjc1 genes, respectively) as shown in Figure 1. Cx30.2 and Cx45 form low-conductance channels (Verheijck et al. 1998; Bukauskas and Verselis 2004; Kreuzberg et al. 2005). These channels integrate thousands of PM cells with various intrinsic frequencies of excitation into one functional unit, which defines the HR in the SAN.

Regulated activity of a nonselective cation channel, the funny current (If) channel is thought to be the PM current in the SAN. The If channel can inwardly conduct both sodium and potassium ions on direct binding of cAMP (DiFrancesco and Tortora 1991). As the membrane potential becomes increasingly hyperpolarized, If increases inward potassium and sodium currents, which causes diastolic depolarization.

The calcium clock is the other driving mechanism behind the autorhythmicity of PM cells within the SAN (Lakatta and DiFrancesco 2009). Increased Ca2+ entry in the sarcoplasmic reticulum increases automaticity because of the effect of [Ca2+]i on the transient inward current carried by the sodium–calcium exchange current (INCX). When these pacemaking mechanisms depolarize the resting membrane potential and reach the threshold voltage, L-type Ca channels (LTCCs) open, and an action potential is fired.

Gene Regulatory Mechanisms for SAN Development and Function

Our insights into mechanisms of CCS development are mostly derived from transgenic mouse models that allow manipulation of CCS genes in vivo (Liang et al. 2013; Sun et al. 2013; Wu et al. 2014a).

Tbx3 (encoding the T-box TF 3) expression is specifically maintained in the SAN anlage all the way to mature SAN where it is required to suppress the working myocardial gene program (Gja1, Gja5, and Scn5a) (Hoogaars et al. 2007; Frank et al. 2012). Tbx3 expression is reinforced by auto-activation in conjunction with activated by AT-rich interactive domain-containing protein 1A (ARID1A). TBX3 also complexes with HDAC3 to suppress Nkx2-5 expression in the SAN (Wu et al. 2014b). Restriction of the SAN boundary to the right sinus horn is ensured by Pitx2 (encoding pituitary homeobox2 protein [PITX2]), which is expressed only in the left sinus horn to suppress SV morphogenesis and gene expression (Mommersteeg et al. 2007b). PITX2 directly suppresses Shox2 (encoding short stature homeobox protein 2 [SHOX2]) and Tbx3 expression (Wang et al. 2010, 2014). A comprehensive transcriptional analysis of mammalian PM cells from laser capture–microdissected SAN tissue from Hcn4-GFP bacterial artificial chromosome (BAC) transgenic mice provided evidence that although PM cells and RA myocytes share core transcriptional machinery, mature PM cells show a unique transcriptome (Vedantham et al. 2015).

TBX5 (or T-box TF 5), a transcriptional activator expressed in the SV and its precursors, regulates expression of the PM genes Bmp4, Shox2, and Tbx3 (Mori et al. 2006; Puskaric et al. 2010). Shox2 expression is crucial for SAN development and is restricted to the SV, where it antagonizes Nkx2-5 expression and activates Hcn4, Isl1, and Tbx3 expression (Blaschke et al. 2007; Espinoza-Lewis et al. 2009, 2011). The absence of Nkx2-5 expression is required for the SV to maintain the identity of PM cells from surrounding atrial cells (Mommersteeg et al. 2007a,b; Espinoza-Lewis et al. 2009, 2011; Ye et al. 2015). Shortly after gastrulation, canonical WNT signaling has been implied to inhibit Nkx2-5 expression, which contributes to activation of the SAN gene program (Bressan et al. 2013).

Initially, insulin gene enhancer protein ISL1 (encoded by Isl1 is a LIM-homeobox TF) is expressed by cardiac progenitor cells to mediate SHF development. But thereafter, Isl1 expression becomes restricted to the SAN where it is maintained until adulthood (Sun et al. 2007; Weinberger et al. 2012; Vedantham et al. 2015). ISL1 acts upstream in the SAN signaling cascade to establish and regulate the PM gene program (Bmp4, Hcn4, Shox2, and Tbx3) (Tessadori et al. 2012; Hoffmann et al. 2013; Liang et al. 2015; Vedantham et al. 2015). A positive feedback loop between ISL1 and SHOX2 (Tessadori et al. 2012) has been implicated with important developmental consequences for SAN formation and the heartbeat.

Clinical Implications

From seminal genome-wide association studies (GWAS), variants in and around important protein-coding genes MYH6 (encoding the α heavy chain subunit of cardiac myosin) (Holm et al. 2011), HCN4 (Veldkamp et al. 2003; Milano et al. 2014), SCN5A (α-subunit of cardiac sodium channel gene) (Benson et al. 2003; Smits et al. 2005), ANK2 (Mohler et al. 2003; Le Scouarnec et al. 2008), and SHOX2 (Hoffmann et al. 2016) have been attributed to sick sinus syndrome susceptibility, familial forms of primary sinus bradycardia, and early-onset atrial fibrillation (AF). Furthermore, a cardiac-specific and highly conserved microRNA, miR-208a, encoded by intron 27 of MYH6 (in both humans and mice), was shown in mice to be necessary for maintenance of normal cardiac conduction (Sunyaev et al. 2001; Callis et al. 2009). Another important large meta-analysis of the associations between single- nucleotide polymorphisms (SNPs) and HR in individuals of European ancestry and Indian-Asian ancestry identified several important loci that were shown to have significant and robust association with HR (den Hoed et al. 2013) (individual SNP associations and nearest-neighboring genes are schematized in Figure 5 below).

THE ATRIOVENTRICULAR CONDUCTION SYSTEM (AVCS)

Anatomy and Primary Function

In the normal heart, a fully mature AVN becomes the principal electrical conduit between the atria and ventricles. This node defines the delay in ventricular activation and can act as a secondary PM if the SAN fails (Fig. 3). The delay of conduction at the AVN is necessary for the sequential contraction of atria and ventricles ensuring optimal hemodynamics (Mazgalev and Tchou 2000).

Figure 3.

Figure 3.

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Molecular and signaling networks for atrioventricular conduction system (AVCS) development. Light green inset shows the definitive atrioventricular node (AVN). Black arrows indicate activation of genes. Blue lines with blunt ends indicate suppression of gene expression by respective transcription factors (TFs). The blue dotted box represents a gene program that shuts down, and the light dotted box indicates a gene program that is activated for maturation of the AVN. The large continuous light green box indicates the role of players in AVN development and function. (BMP2) bone morphogenetic protein 2.

Developmental Origin

The atrioventricular canal (AVC) is the first component to form during heart development, largely derived from the limbs of the cardiac crescent in the E7.5 mouse embryo (Davis et al. 2001; de la Cruz et al. 2001; Aanhaanen et al. 2009; Domínguez et al. 2012). The FHF-derived inflow tract (IFT) of the primary heart tube becomes the major part of the AVC and is highly conserved among vertebrates. In mammals and birds, the AVC gives rise to the AVN, AV ring bundles, and the base of the left ventricle (Aanhaanen et al. 2009; Vicente-Steijn et al. 2011) that has the ability to maintain their PM-like phenotype (that is, spontaneous depolarization and slow conduction) into adulthood. It is around the AVC that the atria, SV, ventricles, and outflow tract subsequently develop. At E9.5 during mouse development, CMs in the primary heart tube exit the cell cycle (Burkhard et al. 2017).

Maintenance of Normal Electrical Activity in AVN

In the AVN, cell–cell coupling is mediated by abundantly expressed Cx30.2 and Cx45 and to a lesser extent by Cx40 (encoded by Gja5 [GJ α5]). Cx30.2 and Cx45 possess higher intrinsic resistance and mediate slower conduction in the nodal regions compared with the VCS or chamber myocardium (Bukauskas and Veteikis 1977; Bukauskas 1982). An overlapping expression pattern of Cx30.2 and Cx40 was found only in part of the distal AVN, AVB, and BBs (Fig. 4). Recently, resident macrophages in the AVN were shown to be electrically coupled to myocardial cells in the AVN and to facilitate electrical conduction in a manner dependent on expression of Gja1 (the gene encoding GJ α1 protein; also known as connexin 43 or Cx43) (Hulsmans et al. 2017).

Figure 4.

Figure 4.

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Molecular signaling networks for ventricular conduction system (VCS) development. The orange inset shows the atrioventricular bundle/bundle branches (AVB/BBs) and the green inset shows the Purkinje fiber network (PFN). Black arrows indicate activation of genes. Blue lines with blunt ends indicate suppression of gene expression by respective transcription factor (TFs). The blue dotted box represents gene program that shuts down. Orange and green dotted boxes indicate gene programs that are activated for maturation of the AVB/BB and PFN, respectively. Large continuous orange and green boxes represent the network of players in AVB/BB and PFN development, respectively. (MAPK) mitogen-activated protein kinase.

Conduction in the AVN may also be slowed due to reduced cellular excitability resulting from decreased numbers of Na+ channels, reduced resting potential that partially inactivates inward currents, or branching of fibers (Spach et al. 2000; Kléber and Rudy 2004). Thus, the AVN protects the ventricular myocardium against the transmission of tachyarrhythmias by filtering high depolarization frequencies during AF (Dobrzynski et al. 2003).

Gene Regulatory Mechanisms for AVC Development and Function

Constitutive or inducible KI-Cre drivers enable precise genetic manipulation in vivo (Stroud et al. 2007; Boogerd et al. 2008; Horsthuis et al. 2009; Aanhaanen et al. 2011; Bakker et al. 2012; Frank et al. 2012; Luna-Zurita et al. 2016; Mohan et al. 2018; Bhattacharyya et al. 2019) to gain mechanistic insights into AVCS development and function.

One of the first genome-wide microarray analyses using an AVN-specific Tbx3-GFP BAC transgenic provided insight into the distinct regulatory sequences and pathways that control the formation of the AVN. This study also revealed that the AVN and working myocardium phenotypes diverge during development with maintenance of distinct functional gene repertoires. In the AVN-specific gene expression profiles, multiple neurotrophic factors and semaphorins were identified pointing toward shared characteristics between nodal and nervous system development (Horsthuis et al. 2009).

The embryonic AVC expresses Bmp2, the gene encoding bone morphogenetic protein 2 (BMP2). BMP2 activates the expression of Tbx2 and Tbx3 genes (encoding the T-box TFs, respectively), which act redundantly to repress the chamber myocardial gene program (Harrelson et al. 2004; Ma et al. 2005; Aanhaanen et al. 2011; Singh et al. 2012) and stimulate the PM and neuronal gene program (Horsthuis et al. 2009; Bakker et al. 2012; Singh et al. 2012). AVC-specific deletion of Bmpr1a (which encodes BMP receptor type 1A) leads to impaired development of the AVN and annulus fibrosus, the structure that provides a conduction barrier between the atria and ventricles (Gaussin et al. 2005; Stroud et al. 2007).

TBX2 and TBX3 interact with Msh Homeobox2 MSX2 (encoded by Msx2 gene) in the AVC to suppress Gja1 expression (Boogerd et al. 2008) in a dose-dependent manner. Myocardium-specific loss of Tbx2 results in ectopic (chamber myocardium-like) connections between the atrium and ventricle that conduct the impulse rapidly through the annulus fibrosus, causing ventricular pre-excitation reminiscent of Wolff–Parkinson–White (WPW) syndrome (Aanhaanen et al. 2011).

The core cardiac TFs (homeobox protein NKX2.5, TBX5, GATA4, and GATA6) also interact with localized TFs such as TBX2 and TBX3 to orchestrate development of the AVC. Heterozygous loss of Tbx5 results in maintenance of an extensive fetal-like AVC phenotype in adult MinK–lacZ mice (Kondo et al. 2003; Moskowitz et al. 2004), suggesting that a finely tuned balance between differentiation-suppressive TBX3 and chamber-program-promoting TBX5 activity controls AVCS homeostasis.

While investigating the mechanisms of proper AV delay establishment, a crucial enhancer, regulated by Tbx5 and Gata4, was shown to be responsible for proper Cx30.2 expression pattern in slower-conducting components of the AVN (Kreuzberg et al. 2006; Munshi et al. 2009). Furthermore, Gata4 heterozygous mice have shortened PR intervals, suggesting that Gata4-dependent regulation of Cx30.2 (and other genes) contributes to slow AV nodal conduction and normal AV delay (Munshi et al. 2009). MyoR was also shown to directly interact with Gata4 to mediate transcriptional repression of the Cx30.2 enhancer required for AVN-specific gene expression (Harris et al. 2015). AVC-specific gene expression is also achieved through GATA4-dependent regulatory switches (Stefanovic et al. 2014). Homozygous deletion of MyoR (a transcriptional repressor that interacts with GATA4) results in prolonged AV conduction and increased expression of Gjd3 (Harris et al. 2015).

TBX20 usually suppresses Tbx2 expression in the chambers (possibly through interfering with BMPs and SMAD-mediated activation of Tbx2 expression), which helps to confine TBX2-dependent AVC formation (Singh et al. 2009). Expression of HEY1 and HEY2 (genes encoding the transcriptional repressors hairy/enhancer-of-split related with YRPW motif proteins 1 and 2; HEY1 and HEY2, respectively) is activated by Notch signaling and inhibits BMP2 and TBX2 expression (Rutenberg et al. 2006; Kokubo et al. 2007) in a negative feedback loop (Rutenberg et al. 2006). Ectopic Notch activation in the developing myocardium leads to AVC mispatterning, formation of accessory conduction pathways, and ventricular pre-excitation. Conversely, inhibition of Notch signaling leads to a hypoplastic AVN (Rentschler et al. 2011). GATA4 and GATA6 act in concert with SMAD proteins and nuclear BMP signaling effectors to recruit histone acetyltransferases, thereby driving the AVC gene program. Conversely, GATA4 and GATA6 recruit HEY1 or HEY2 and histone deacetylases (HDACs) in the chambers, resulting in repression of the AVC gene program. Gata6−/− mice show prolonged AV conduction and a hypoplastic AVN (Liu et al. 2015). In mice, WNT loss-of-function leads to progressive loss of the AVC phenotype during late gestation, which by the time of birth results in the near absence of Tbx3+/Gja5−AVC myocardium (Gillers et al. 2015).

Clinical Implications

One of the early studies to investigate genetic causes of progressive heart block and AV nodal dysfunction identified human mutations in Nkx2-5 gene (Pashmforoush et al. 2004). Careful clinical examination of patients with Holt–Oram syndrome (HOS), an autosomal-dominant condition characterized by a familial congenital heart defects and pre-axial radial ray upper limb defects, have identified almost 37 associative truncation mutations in TBX5 in more than 70% of cases. (Mori and Bruneau 2004). A curated list of important TFs and signaling pathways implicated in human CCS function has been summarized previously (van Eif et al. 2018). Defects in the PRKAG2 gene, encoding the AMP-activated protein kinase (AMPK) γ2 regulatory subunit have been associated with a cardiac syndrome triad consisting of WPW, conduction system disease, and hypertrophic cardiomyopathy (Liu et al. 2013).

Meta-analyses of GWAS from European individuals reported multiple PR interval loci inclusive of previously described GWAS variants (Pfeufer et al. 2010; van Setten et al. 2018, 2019). Two additional studies on African-American and Asian ancestries identified two significant and unique PR interval–associated loci (Smith et al. 2011; Sano et al. 2014; Christophersen et al. 2017). In the last decade, GWAS and exposome-wide association studies (ExWAS) have identified 25 AF genetic loci from large-scale combined ancestry analysis, many of which showed nominal association with the PR interval (Low et al. 2017; van Setten et al. 2019). An updated list of the significantly associated genetic loci with multiple human atrial rhythm parameters and AF is shown in Figure 5.

THE VCS

Anatomy and Primary Function

The sole electrical bridge between the atrial and ventricular myocardium is composed of the fast-conducting AVB, which is connected to the AVN and continues through the crest of the ventricular septum. It propagates the electrical impulse to the BBs and terminates at the PFN, which activates the ventricular myocardium.

Maintenance of Normal Electrical Activity in VCS

The postnatal VCS expresses genes for fast conduction (Gja5 and Scn5a), PM properties (Hcn4), neuronal neurofilament proteins (Nefm and Nefl), and the axonal glycoprotein contactin 2 (Cntn2) (Gorza and Vitadello 1989; Moskowitz et al. 2004; Pallante et al. 2010; Arnolds et al. 2012; Liang et al. 2013).

Hcn4 is progressively turned on in the VCS in fetal period, whereas Cntn2 expression is activated perinatally, indicating that the VCS undergoes further maturation after birth (Pallante et al. 2010; Beyer et al. 2011; Bhattacharyya et al. 2017).

Cx40 is abundantly expressed in the AVB, BBs, and subendocardial network of PFs, that is, structures with high conduction velocities (Beyer et al. 1989; Gourdie et al. 1993; Coppen et al. 1999). In the AVB and BBs, Cx40 and Cx45 expression overlaps, and the expression of Cx45 decreases at the transition from BBs to PFN with increased expression of Cx43 (for review, see Lo 2000). From the Purkinje terminals, the excitation is transferred to the ventricular myocardium presumably through Cx43 GJs.

AVB AND BBs

Developmental Origin

The AVB is conserved across mammals and birds but absent in fish, amphibians, and reptiles—except for crocodilians (Jensen et al. 2018). The AVB develops from the ventricular septal part of the Tbx3+ primary interventricular ring, and the BBs are derived from the Tbx3+ subendocardial trabecules, which develop into the ventricular septum (Wessels et al. 1992; Hoogaars et al. 2004). Lineage-tracing studies indicate that the AVB and right BB originate from CM progenitors that express SHF markers (Aanhaanen et al. 2010; Devine et al. 2014). It is by virtue of high expression of Scn5a in the AVB during initial development followed by the induction of Gja5 expression during the fetal period that AVB and BBs attain a fast conduction phenotype (Davis et al. 1995; Moskowitz et al. 2004; Yoo et al. 2006; Remme et al. 2009; Arnolds et al. 2012).

Gene Regulatory Mechanisms for AVB and BB Development and Function

An intricate tissue-specific gene regulatory network (GRN) composed of several TFs—NKX2.5, TBX3, TBX5, DNA-binding protein inhibitor ID2 (encoded by Id2), ETS translocation variant 1 (ETV1, encoded by Etv1), homeodomain-only protein (HOPX, encoded by Hopx), TF SP4 (encoded by Sp4), and iroquois-class homeodomain protein IRX3 (encoded by Irx3) have been described (Nguyêñ-Trân et al. 2000; Moskowitz et al. 2004; 2007; Ismat et al. 2005; Zhang et al. 2011; Arnolds et al. 2012; Bakker et al. 2012; Shekhar et al. 2016).

In the early AVB anlage, TBX3 suppresses transcription of fast conduction genes (e.g., Gja1 and Gja5), inhibits cell proliferation, and stimulates a slowly conducting nodal gene program. Although Gja1 expression remains suppressed in the AVB and BBs, expression of Gja5 (Bruneau et al. 2001; Hoogaars et al. 2007) is initiated in the fetal AVB. In contrast, Scn5a (van den Boogaard et al. 2012) is highly expressed in both the AVB and BBs from early development. This could be explained by competition between the transcriptional activator TBX5 and the transcriptional repressor TBX3 for interaction with Nkx2-5 to bind common regulatory elements (Bakker et al. 2008; van den Boogaard et al. 2012).

TBX5 regulates the development and homeostasis of the AVB and BBs (Moskowitz et al. 2007; Arnolds et al. 2012) by directly activating expression of Gja5 and Scn5a in the AVB ensuring rapid conduction. Nkx2-5+/− mice develop AVB hypoplasia and AV block, which is largely rescued by Prox1 (gene encoding for prospero homeobox protein 1 [PROX1]) haploinsufficiency, possibly through alleviating transcriptional repression of Nkx2-5 mediated by HDAC3 and/or PROX1 (Risebro et al. 2012).

THE PURKINJE FIBER NETWORK

Developmental Origin

The PFN is derived from cells of the early ventricular chamber myocardium that initiate the expression of Bmp10, Gja5, Irx3, and Nppa as soon as they differentiate from the primary heart tube (mouse E9 stage) (Delorme et al. 1997; Christoffels et al. 2000a,b; Chen et al. 2004; Miquerol et al. 2010; Zhang et al. 2011). Irreversible genetic labeling of Gja5+ cells during mouse development has revealed that definitive VCS is established at mid- to late-fetal stages (Miquerol et al. 2010). In mammals and birds, the trabecular layer develops into a specialized PFN (Jensen et al. 2018).

Gene Regulatory Mechanisms for PFN Development and Function

Development of genetic labeling tools for VCS led to important findings in VCS anatomy and biology (Rentschler et al. 2001; Poliak et al. 2003; Pallante et al. 2010; Arnolds and Moskowitz 2011; Bhattacharyya et al. 2017).

Endocardial Notch signaling stimulates expression of Bmp10 in trabecular myocardium. This event suppresses cell-cycle inhibitor gene expression to ensure maintenance of the trabecular zone throughout development (Chen et al. 2004). Additionally, endocardial Notch signaling stimulates expression of Nrg1 (neuregulin 1) to induce fast conduction genes (e.g., Gja5, Irx3, Nkx2-5, and Scn5a) and the transgenic reporter CCS–lacZ, via RAS–mitogen-activated protein kinase (MAPK) signaling through ETV1 (Rentschler et al. 2002; Shekhar et al. 2016). Another study identified the TF ETV1 to be highly abundant in pectinated atrial myocardium (PAM) and VCS myocytes. Expression of Etv1 subsequently becomes restricted to the VCS in a pattern that resembles that of Gja5, a target gene of ETV1. Etv1−/− mice have a hypoplastic VCS with reduced expression of Gja5, Nkx2-5, and Scn5a along with a prolonged P wave, PR interval, and QRS duration (Shekhar et al. 2016). Postnatally, the trabecular zone remodels to become the mature PFN, which is only a few cells thick. This remodeling process requires IRX3, ETV1, and normal levels of NKX2.5, but the exact mechanism is not yet clear (Meysen et al. 2007; Zhang et al. 2011; Kim et al. 2016; Shekhar et al. 2016).

Postnatally, expression of Irx3 is required for VCS maturation by inducing Gja5 expression and Gja1 suppression in the VCS. Irx3−/− mice display ventricular conduction slowing, QRS prolongation, and ventricular tachyarrhythmias (Zhang et al. 2011). Expression of Irx3, Irx5, and Tbx5 is higher in the trabecular region on the endocardial side. These TFs are involved in the regulation of Gja5, Scn5a, and genes related to conduction and repolarization (Costantini et al. 2005; Zhang et al. 2011; Arnolds et al. 2012; Gaborit et al. 2012; Koizumi et al. 2016). HEY2 is a transcriptional repressor that suppresses Gja5, Nppa, and Scn5a expression in the compact zone and activates expression of Kcnd2 and Kcnip2 in the subepicardial domain (Koibuchi and Chin 2007; Xin et al. 2007; Bezzina et al. 2013; Veerman et al. 2017).

Nkx2.5 increases Cx40 expression directly by binding to the Cx40 promoter and indirectly by activating HOPX in the AVB, BB, and PFN (Ismat et al. 2005). HF-1b (Zinc finger protein) is also expressed within the VCS and impacts its electrical properties by simultaneously up-regulating Cx40 and down-regulating the minK potassium channel (Hewett et al. 2005).

Clinical Implications

GWAS of human genes associated with ECG parameters like PR interval, QRS interval durations, ventricular fibrillation, and conduction abnormalities (BB block and heart block) identified variants in ion channels and important TFs (TBX3, TBX5, IRX3, and ETV1) (van den Boogaard et al. 2012, 2014; Koizumi et al. 2016; Shekhar et al. 2016). Short QT syndrome (SQTS) and Brugada-like ECG has been associated with gain-of-function mutations in potassium channel genes, loss-of-function mutations in relevant calcium channel genes, and HEY2 with associated ion channel (SCN5A-SCN10A) patterning defects (Hong et al. 2012; Kim et al. 2012; Bezzina et al. 2013; Veerman et al. 2017). A meta-analysis of European individuals revealed multiple interesting and relevant loci that were significantly associated with QRS duration (Sotoodehnia et al. 2010). Another meta-analysis identified two additional novel locus-trait associations: NFKB1 for QT interval and ATP2A2 for QRS duration (van Setten et al. 2019). An illustration of the significantly associated genetic loci with human QRS duration and QT interval is shown in Figure 5.

CURRENT CCS DISEASE MODELS, DRUG TESTING, AND POTENTIAL FUTURE THERAPIES

Catheter ablation and device implantation are the most frequently used therapies for managing cardiac arrhythmias. Although electronic PMs have many advanced features, such as an extended battery life and advanced HR adaptation algorithms, their use is limited in specific patient populations. Thus, the need for creating a biological PM remains an important and exciting research challenge.

Efforts to create a biological PM have focused on three strategies: introduction of specific ion channels into CMs by direct gene transfer, expressing ion channel in non-CMs followed by cell fusion to native CMs in situ, and the introduction of stem cells that have been previously differentiated into CMs (Miake et al. 2002; Potapova et al. 2004; Cho et al. 2007; Plotnikov et al. 2007). To recapitulate additional features of PM cells, embryonic stem cells (ESCs) were differentiated in vitro into PM-like CMs and then introduced directly into the heart of pigs or guinea pigs (Kehat et al. 2004; Xue et al. 2005; Tse et al. 2006).

In the last decade, using cues from developmental biology, significant advances have been made in our understanding of cardiac differentiation from mouse and human ESCs (m/hESCs) and induced pluripotent stem cells (m/hiPSCs) to induced CMs (Yang et al. 2008; Kattman et al. 2011; Lee et al. 2017). A recent study reported the generation of an hiPSC double reporter to isolate lineage-specific cardiac subpopulations, including the FHF, epicardial, SHF, and endothelial lineages (Zhang et al. 2019). Taken together, these studies establish an in vitro platform for human disease modeling and potential drug testing. However, current differentiation protocols do not selectively promote specific CCS-like CM subtypes. To address this challenge, recent work showed specification of functional SAN-like PM cells (SANLPCs) from hPSCs on stage-specific activation of BMP and retinoic acid, and inhibition of FGF (fibroblast growth factor) signaling pathways (Protze et al. 2017).

CONCLUSION

The CCS is composed of a set of specialized CMs that generate and propagate the electrical impulse required for cardiac contraction. The SAN acts as the PM to initiate cardiac rhythm (Rubart and Zipes 2005) and SAN damage often requires artificial PM implantation in human patients. Alternatively, a biological PM could be advantageous in particular clinical situations. Recently, differentiation of hPSCs into SAN-like PM cells (SANLPCs) (Protze et al. 2017) and direct reprogramming of fibroblasts have emerged as viable strategies to generate a biological PM using a developmental biology-guided approach (Fernandez-Perez et al. 2019). However, neither is optimal owing to a general lack of knowledge regarding CCS GRNs. These observations emphasize the need for an improved roadmap for CCS regeneration therapies, an area of intense ongoing investigation.

ACKNOWLEDGMENTS

We apologize to the many investigators whose work could not be cited because of space constraints. We thank all members of the Munshi Laboratory for insightful discussions. This work was supported by the AHA (17PRE33670730 to S.B.), NIH (HL136604, HL133642, and HL135217 to N.V.M), the Burroughs Wellcome Fund (1009838 to N.V.M.), the March of Dimes Foundation (#5-FY13-203 to N.V.M.), and the Department of Defense (PR172060 to N.V.M.).

Footnotes

Editors: Benoit G. Bruneau and Paul R. Riley

Additional Perspectives on Heart Development and Disease available at www.cshperspectives.org

REFERENCES

  1. Aanhaanen WT, Brons JF, Domínguez JN, Rana MS, Norden J, Airik R, Wakker V, de Gier-de Vries C, Brown NA, Kispert A, et al. 2009. The Tbx2+ primary myocardium of the atrioventricular canal forms the atrioventricular node and the base of the left ventricle. Circ Res 104: 1267–1274. 10.1161/CIRCRESAHA.108.192450 [DOI] [PubMed] [Google Scholar]
  2. Aanhaanen WT, Mommersteeg MT, Norden J, Wakker V, de Gier-de Vries C, Anderson RH, Kispert A, Moorman AF, Christoffels VM. 2010. Developmental origin, growth, and three-dimensional architecture of the atrioventricular conduction axis of the mouse heart. Circ Res 107: 728–736. 10.1161/CIRCRESAHA.110.222992 [DOI] [PubMed] [Google Scholar]
  3. Aanhaanen WT, Boukens BJ, Sizarov A, Wakker V, de Gier-de Vries C, van Ginneken AC, Moorman AF, Coronel R, Christoffels VM. 2011. Defective Tbx2-dependent patterning of the atrioventricular canal myocardium causes accessory pathway formation in mice. J Clin Invest 121: 534–544. 10.1172/JCI44350 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Arnolds DE, Moskowitz IP. 2011. Inducible recombination in the cardiac conduction system of minK: CreERT2 BAC transgenic mice. Genesis 49: 878–884. 10.1002/dvg.20759 [DOI] [PubMed] [Google Scholar]
  5. Arnolds DE, Liu F, Fahrenbach JP, Kim GH, Schillinger KJ, Smemo S, McNally EM, Nobrega MA, Patel VV, Moskowitz IP. 2012. TBX5 drives Scn5a expression to regulate cardiac conduction system function. J Clin Invest 122: 2509–2518. 10.1172/JCI62617 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bakker ML, Boukens BJ, Mommersteeg MT, Brons JF, Wakker V, Moorman AF, Christoffels VM. 2008. Transcription factor Tbx3 is required for the specification of the atrioventricular conduction system. Circ Res 102: 1340–1349. 10.1161/CIRCRESAHA.107.169565 [DOI] [PubMed] [Google Scholar]
  7. Bakker ML, Boink GJ, Boukens BJ, Verkerk AO, van den Boogaard M, den Haan AD, Hoogaars WM, Buermans HP, de Bakker JM, Seppen J, et al. 2012. T-box transcription factor TBX3 reprogrammes mature cardiac myocytes into pacemaker-like cells. Cardiovasc Res 94: 439–449. 10.1093/cvr/cvs120 [DOI] [PubMed] [Google Scholar]
  8. Benson DW, Wang DW, Dyment M, Knilans TK, Fish FA, Strieper MJ, Rhodes TH, George AL Jr. 2003. Congenital sick sinus syndrome caused by recessive mutations in the cardiac sodium channel gene (SCN5A). J Clin Invest 112: 1019–1028. 10.1172/JCI200318062 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Beyer EC, Kistler J, Paul DL, Goodenough DA. 1989. Antisera directed against connexin43 peptides react with a 43-kD protein localized to gap junctions in myocardium and other tissues. J Cell biol 108: 595–605. 10.1083/jcb.108.2.595 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Beyer S, Kelly RG, Miquerol L. 2011. Inducible Cx40-Cre expression in the cardiac conduction system and arterial endothelial cells. Genesis 49: 83–91. 10.1002/dvg.20687 [DOI] [PubMed] [Google Scholar]
  11. Bezzina CR, Barc J, Mizusawa Y, Remme CA, Gourraud JB, Simonet F, Verkerk AO, Schwartz PJ, Crotti L, Dagradi F, et al. 2013. Common variants at SCN5A-SCN10A and HEY2 are associated with Brugada syndrome, a rare disease with high risk of sudden cardiac death. Nat Genet 45: 1044–1049. 10.1038/ng.2712 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Bhattacharyya S, Bhakta M, Munshi NV. 2017. Phenotypically silent Cre recombination within the postnatal ventricular conduction system. PLoS ONE 12: e0174517 10.1371/journal.pone.0174517 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Bhattacharyya S, Duan J, Wang L, Li B, Bhakta M, Fernandez-Perez A, Hon GC, Munshi NV. 2019. Using Gjd3-CreEGFP mice to examine atrioventricular node morphology and composition. Sci Rep 9: 2106 10.1038/s41598-019-38683-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Blaschke RJ, Hahurij ND, Kuijper S, Just S, Wisse LJ, Deissler K, Maxelon T, Anastassiadis K, Spitzer J, Hardt SE, et al. 2007. Targeted mutation reveals essential functions of the homeodomain transcription factor Shox2 in sinoatrial and pacemaking development. Circulation 115: 1830–1838. 10.1161/CIRCULATIONAHA.106.637819 [DOI] [PubMed] [Google Scholar]
  15. Boogerd KJ, Wong LY, Christoffels VM, Klarenbeek M, Ruijter JM, Moorman AF, Barnett P. 2008. Msx1 and Msx2 are functional interacting partners of T-box factors in the regulation of connexin43. Cardiovasc Res 78: 485–493. 10.1093/cvr/cvn049 [DOI] [PubMed] [Google Scholar]
  16. Bressan M, Liu G, Mikawa T. 2013. Early mesodermal cues assign avian cardiac pacemaker fate potential in a tertiary heart field. Science 340: 744–748. 10.1126/science.1232877 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Bruneau BG, Nemer G, Schmitt JP, Charron F, Robitaille L, Caron S, Conner DA, Gessler M, Nemer M, Seidman CE, et al. 2001. A murine model of Holt–Oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis and disease. Cell 106: 709–721. 10.1016/S0092-8674(01)00493-7 [DOI] [PubMed] [Google Scholar]
  18. Bukauskas F. 1982. Electrophysiology of the normal-to-hypoxic transition zone. Circ Res 51: 321–329. 10.1161/01.RES.51.3.321 [DOI] [PubMed] [Google Scholar]
  19. Bukauskas FF, Veteikis RP. 1977. Passive electrical properties of the atrioventricular region of the rabbit heart. Biofizika 22: 499–504. [PubMed] [Google Scholar]
  20. Bukauskas FF, Verselis VK. 2004. Gap junction channel gating. Biochim Biophys Acta 1662: 42–60. 10.1016/j.bbamem.2004.01.008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Burkhard S, van Eif V, Garric L, Christoffels VM, Bakkers J. 2017. On the evolution of the cardiac pacemaker. J Cardiovasc Dev Dis 4: 4 10.3390/jcdd4020004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Callis TE, Pandya K, Seok HY, Tang RH, Tatsuguchi M, Huang ZP, Chen JF, Deng Z, Gunn B, Shumate J, et al. 2009. microRNA-208a is a regulator of cardiac hypertrophy and conduction in mice. J Clin Invest 119: 2772–2786. 10.1172/JCI36154 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Chen H, Shi S, Acosta L, Li W, Lu J, Bao S, Chen Z, Yang Z, Schneider MD, Chien KR, et al. 2004. BMP10 is essential for maintaining cardiac growth during murine cardiogenesis. Development 131: 2219–2231. 10.1242/dev.01094 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Cho HC, Kashiwakura Y, Marbán E. 2007. Creation of a biological pacemaker by cell fusion. Circ Res 100: 1112–1115. 10.1161/01.RES.0000265845.04439.78 [DOI] [PubMed] [Google Scholar]
  25. Christoffels VM, Habets PE, Franco D, Campione M, de Jong F, Lamers WH, Bao ZZ, Palmer S, Biben C, Harvey RP, et al. 2000a. Chamber formation and morphogenesis in the developing mammalian heart. Dev Biol 223: 266–278. 10.1006/dbio.2000.9753 [DOI] [PubMed] [Google Scholar]
  26. Christoffels VM, Keijser AG, Houweling AC, Clout DE, Moorman AF. 2000b. Patterning the embryonic heart: identification of five mouse Iroquois homeobox genes in the developing heart. Dev Biol 224: 263–274. 10.1006/dbio.2000.9801 [DOI] [PubMed] [Google Scholar]
  27. Christoffels VM, Mommersteeg MT, Trowe MO, Prall OW, de Gier-de Vries C, Soufan AT, Bussen M, Schuster-Gossler K, Harvey RP, Moorman AF, et al. 2006. Formation of the venous pole of the heart from an Nkx2-5-negative precursor population requires Tbx18. Circ Res 98: 1555–1563. 10.1161/01.RES.0000227571.84189.65 [DOI] [PubMed] [Google Scholar]
  28. Christoffels VM, Smits GJ, Kispert A, Moorman AF. 2010. Development of the pacemaker tissues of the heart. Circ Res 106: 240–254. 10.1161/CIRCRESAHA.109.205419 [DOI] [PubMed] [Google Scholar]
  29. Christophersen IE, Magnani JW, Yin X, Barnard J, Weng LC, Arking DE, Niemeijer MN, Lubitz SA, Avery CL, Duan Q, et al. 2017. Fifteen genetic loci associated with the electrocardiographic P wave. Circ Cardiovasc Genet 10: e001667. 10.1161/CIRCGENETICS.116.001667 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Coppen SR, Kodama I, Boyett MR, Dobrzynski H, Takagishi Y, Honjo H, Yeh HI, Severs NJ. 1999. Connexin45, a major connexin of the rabbit sinoatrial node, is co-expressed with connexin43 in a restricted zone at the nodal-crista terminalis border. J Histochem Cytochem 47: 907–918. 10.1177/002215549904700708 [DOI] [PubMed] [Google Scholar]
  31. Costantini DL, Arruda EP, Agarwal P, Kim KH, Zhu Y, Zhu W, Lebel M, Cheng CW, Park CY, Pierce SA, et al. 2005. The homeodomain transcription factor Irx5 establishes the mouse cardiac ventricular repolarization gradient. Cell 123: 347–358. 10.1016/j.cell.2005.08.004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Davis LM, Rodefeld ME, Green K, Beyer EC, Saffitz JE. 1995. Gap junction protein phenotypes of the human heart and conduction system. J Cardiovasc Electrophysiol 6: 813–822. 10.1111/j.1540-8167.1995.tb00357.x [DOI] [PubMed] [Google Scholar]
  33. Davis DL, Edwards AV, Juraszek AL, Phelps A, Wessels A, Burch JB. 2001. A GATA-6 gene heart-region-specific enhancer provides a novel means to mark and probe a discrete component of the mouse cardiac conduction system. Mech Dev 108: 105–119. 10.1016/S0925-4773(01)00500-7 [DOI] [PubMed] [Google Scholar]
  34. de la Cruz MV, Markwald RR, Krug EL, Rumenoff L, Sánchez Gómez C, Sadowinski S, Galicia TD, Gómez F, Salazar García M, Villavicencio Guzmán L, et al. 2001. Living morphogenesis of the ventricles and congenital pathology of their component parts. Cardiol Young 11: 588–600. 10.1017/S1047951101000932 [DOI] [PubMed] [Google Scholar]
  35. Delorme B, Dahl E, Jarry-Guichard T, Briand JP, Willecke K, Gros D, Théveniau-Ruissy M. 1997. Expression pattern of connexin gene products at the early developmental stages of the mouse cardiovascular system. Circ Res 81: 423–437. 10.1161/01.RES.81.3.423 [DOI] [PubMed] [Google Scholar]
  36. den Hoed M, Eijgelsheim M, Esko T, Brundel BJ, Peal DS, Evans DM, Nolte IM, Segrè AV, Holm H, Handsaker RE, et al. 2013. Identification of heart rate-associated loci and their effects on cardiac conduction and rhythm disorders. Nat Genet 45: 621–631. 10.1038/ng.2610 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Devine WP, Wythe JD, George M, Koshiba-Takeuchi K, Bruneau BG. 2014. Early patterning and specification of cardiac progenitors in gastrulating mesoderm. eLife 3: e03848 10.7554/eLife.03848 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. DiFrancesco D, Tortora P. 1991. Direct activation of cardiac pacemaker channels by intracellular cyclic AMP. Nature 351: 145–147. 10.1038/351145a0 [DOI] [PubMed] [Google Scholar]
  39. Dobrzynski H, Nikolski VP, Sambelashvili AT, Greener ID, Yamamoto M, Boyett MR, Efimov IR. 2003. Site of origin and molecular substrate of atrioventricular junctional rhythm in the rabbit heart. Circ Res 93: 1102–1110. 10.1161/01.RES.0000101913.95604.B9 [DOI] [PubMed] [Google Scholar]
  40. Domínguez JN, Meilhac SM, Bland YS, Buckingham ME, Brown NA. 2012. Asymmetric fate of the posterior part of the second heart field results in unexpected left/right contributions to both poles of the heart. Circ Res 111: 1323–1335. 10.1161/CIRCRESAHA.112.271247 [DOI] [PubMed] [Google Scholar]
  41. Espinoza-Lewis RA, Yu L, He F, Liu H, Tang R, Shi J, Sun X, Martin JF, Wang D, Yang J, et al. 2009. Shox2 is essential for the differentiation of cardiac pacemaker cells by repressing Nkx2-5. Dev Biol 327: 376–385. 10.1016/j.ydbio.2008.12.028 [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Espinoza-Lewis RA, Liu H, Sun C, Chen C, Jiao K, Chen Y. 2011. Ectopic expression of Nkx2.5 suppresses the formation of the sinoatrial node in mice. Dev Biol 356: 359–369. 10.1016/j.ydbio.2011.05.663 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Fernandez-Perez A, Sathe AA, Bhakta M, Leggett K, Xing C, Munshi NV. 2019. Hand2 selectively reorganizes chromatin accessibility to induce pacemaker-like transcriptional reprogramming. Cell Rep 27: 2354–2369.e7. 10.1016/j.celrep.2019.04.077 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Frank DU, Carter KL, Thomas KR, Burr RM, Bakker ML, Coetzee WA, Tristani-Firouzi M, Bamshad MJ, Christoffels VM, Moon AM. 2012. Lethal arrhythmias in Tbx3-deficient mice reveal extreme dosage sensitivity of cardiac conduction system function and homeostasis. Proc Natl Acad Sci 109: E154–E163. 10.1073/pnas.1115165109 [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Gaborit N, Sakuma R, Wylie JN, Kim KH, Zhang SS, Hui CC, Bruneau BG. 2012. Cooperative and antagonistic roles for Irx3 and Irx5 in cardiac morphogenesis and postnatal physiology. Development 139: 4007–4019. 10.1242/dev.081703 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Garcia-Frigola C, Shi Y, Evans SM. 2003. Expression of the hyperpolarization-activated cyclic nucleotide-gated cation channel HCN4 during mouse heart development. Gene Expr Patterns 3: 777–783. 10.1016/S1567-133X(03)00125-X [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Gaussin V, Morley GE, Cox L, Zwijsen A, Vance KM, Emile L, Tian Y, Liu J, Hong C, Myers D, et al. 2005. Alk3/Bmpr1a receptor is required for development of the atrioventricular canal into valves and annulus fibrosus. Circ Res 97: 219–226. 10.1161/01.RES.0000177862.85474.63 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Gillers BS, Chiplunkar A, Aly H, Valenta T, Basler K, Christoffels VM, Efimov IR, Boukens BJ, Rentschler S. 2015. Canonical wnt signaling regulates atrioventricular junction programming and electrophysiological properties. Circ Res 116: 398–406. 10.1161/CIRCRESAHA.116.304731 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Gordan R, Gwathmey JK, Xie LH. 2015. Autonomic and endocrine control of cardiovascular function. World J Cardiol 7: 204–214. 10.4330/wjc.v7.i4.204 [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Gorza L, Vitadello M. 1989. Distribution of conduction system fibers in the developing and adult rabbit heart revealed by an antineurofilament antibody. Circ Res 65: 360–369. 10.1161/01.RES.65.2.360 [DOI] [PubMed] [Google Scholar]
  51. Gourdie RG, Severs NJ, Green CR, Rothery S, Germroth P, Thompson RP. 1993. The spatial distribution and relative abundance of gap-junctional connexin40 and connexin43 correlate to functional properties of components of the cardiac atrioventricular conduction system. J Cell Sci 105: 985–991. [DOI] [PubMed] [Google Scholar]
  52. Harrelson Z, Kelly RG, Goldin SN, Gibson-Brown JJ, Bollag RJ, Silver LM, Papaioannou VE. 2004. Tbx2 is essential for patterning the atrioventricular canal and for morphogenesis of the outflow tract during heart development. Development 131: 5041–5052. 10.1242/dev.01378 [DOI] [PubMed] [Google Scholar]
  53. Harris JP, Bhakta M, Bezprozvannaya S, Wang L, Lubczyk C, Olson EN, Munshi NV. 2015. MyoR modulates cardiac conduction by repressing Gata4. Mol Cell Biol 35: 649–661. 10.1128/MCB.00860-14 [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Hewett KW, Norman LW, Sedmera D, Barker RJ, Justus C, Zhang J, Kubalak SW, Gourdie RG. 2005. Knockout of the neural and heart expressed gene HF-1b results in apical deficits of ventricular structure and activation. Cardiovasc Res 67: 548–560. 10.1016/j.cardiores.2005.04.002 [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Hoffmann S, Berger IM, Glaser A, Bacon C, Li L, Gretz N, Steinbeisser H, Rottbauer W, Just S, Rappold G. 2013. Islet1 is a direct transcriptional target of the homeodomain transcription factor Shox2 and rescues the Shox2-mediated bradycardia. Basic Res Cardiol 108: 339 10.1007/s00395-013-0339-z [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Hoffmann S, Clauss S, Berger IM, Weiß B, Montalbano A, Röth R, Bucher M, Klier I, Wakili R, Seitz H, et al. 2016. Coding and non-coding variants in the SHOX2 gene in patients with early-onset atrial fibrillation. Basic Res Cardiol 111: 36 10.1007/s00395-016-0557-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Holm H, Gudbjartsson DF, Sulem P, Masson G, Helgadottir HT, Zanon C, Magnusson OT, Helgason A, Saemundsdottir J, Gylfason A, et al. 2011. A rare variant in MYH6 is associated with high risk of sick sinus syndrome. Nat Genet 43: 316–320. 10.1038/ng.781 [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Hong K, Hu J, Yu J, Brugada R. 2012. Concomitant Brugada-like and short QT electrocardiogram linked to SCN5A mutation. Eur J Hum Genet 20: 1189–1192. 10.1038/ejhg.2012.63 [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Hoogaars WM, Tessari A, Moorman AF, de Boer PA, Hagoort J, Soufan AT, Campione M, Christoffels VM. 2004. The transcriptional repressor Tbx3 delineates the developing central conduction system of the heart. Cardiovasc Res 62: 489–499. 10.1016/j.cardiores.2004.01.030 [DOI] [PubMed] [Google Scholar]
  60. Hoogaars WM, Engel A, Brons JF, Verkerk AO, de Lange FJ, Wong LY, Bakker ML, Clout DE, Wakker V, Barnett P, et al. 2007. Tbx3 controls the sinoatrial node gene program and imposes pacemaker function on the atria. Genes Dev 21: 1098–1112. 10.1101/gad.416007 [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Horsthuis T, Buermans HP, Brons JF, Verkerk AO, Bakker ML, Wakker V, Clout DE, Moorman AF, ’t Hoen PA, Christoffels VM. 2009. Gene expression profiling of the forming atrioventricular node using a novel Tbx3-based node-specific transgenic reporter. Circ Res 105: 61–69. 10.1161/CIRCRESAHA.108.192443 [DOI] [PubMed] [Google Scholar]
  62. Hulsmans M, Clauss S, Xiao L, Aguirre AD, King KR, Hanley A, Hucker WJ, Wülfers EM, Seemann G, Courties G, et al. 2017. Macrophages facilitate electrical conduction in the heart. Cell 169: 510–522.e20. 10.1016/j.cell.2017.03.050 [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Ismat FA, Zhang M, Kook H, Huang B, Zhou R, Ferrari VA, Epstein JA, Patel VV. 2005. Homeobox protein Hop functions in the adult cardiac conduction system. Circ Res 96: 898–903. 10.1161/01.RES.0000163108.47258.f3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Jensen B, Boukens BJ, Crossley DA II, Conner J, Mohan RA, van Duijvenboden K, Postma AV, Gloschat CR, Elsey RM, Sedmera D, et al. 2018. Specialized impulse conduction pathway in the alligator heart. eLife 7: e32120. doi:10.7554/eLife.32120 [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Kamino K, Hirota A, Fujii S. 1981. Localization of pacemaking activity in early embryonic heart monitored using voltage-sensitive dye. Nature 290: 595–597. 10.1038/290595a0 [DOI] [PubMed] [Google Scholar]
  66. Kattman SJ, Witty AD, Gagliardi M, Dubois NC, Niapour M, Hotta A, Ellis J, Keller G. 2011. Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell 8: 228–240. 10.1016/j.stem.2010.12.008 [DOI] [PubMed] [Google Scholar]
  67. Kehat I, Khimovich L, Caspi O, Gepstein A, Shofti R, Arbel G, Huber I, Satin J, Itskovitz-Eldor J, Gepstein L. 2004. Electromechanical integration of cardiomyocytes derived from human embryonic stem cells. Nat Biotechnol 22: 1282–1289. 10.1038/nbt1014 [DOI] [PubMed] [Google Scholar]
  68. Kim JW, Hong KW, Go MJ, Kim SS, Tabara Y, Kita Y, Tanigawa T, Cho YS, Han BG, Oh B. 2012. A common variant in SLC8A1 is associated with the duration of the electrocardiographic QT interval. Am J Hum Genet 91: 180–184. 10.1016/j.ajhg.2012.05.019 [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Kim KH, Rosen A, Hussein SM, Puviindran V, Korogyi AS, Chiarello C, Nagy A, Hui CC, Backx PH. 2016. Irx3 is required for postnatal maturation of the mouse ventricular conduction system. Sci Rep 6: 19197 10.1038/srep19197 [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Kléber AG, Rudy Y. 2004. Basic mechanisms of cardiac impulse propagation and associated arrhythmias. Physiol Rev 84: 431–488. 10.1152/physrev.00025.2003 [DOI] [PubMed] [Google Scholar]
  71. Koibuchi N, Chin MT. 2007. CHF1/Hey2 plays a pivotal role in left ventricular maturation through suppression of ectopic atrial gene expression. Circ Res 100: 850–855. 10.1161/01.RES.0000261693.13269.bf [DOI] [PubMed] [Google Scholar]
  72. Koizumi A, Sasano T, Kimura W, Miyamoto Y, Aiba T, Ishikawa T, Nogami A, Fukamizu S, Sakurada H, Takahashi Y, et al. 2016. Genetic defects in a His-Purkinje system transcription factor, IRX3, cause lethal cardiac arrhythmias. Eur Heart J 37: 1469–1475. 10.1093/eurheartj/ehv449 [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Kokubo H, Tomita-Miyagawa S, Hamada Y, Saga Y. 2007. Hesr1 and Hesr2 regulate atrioventricular boundary formation in the developing heart through the repression of Tbx2. Development 134: 747–755. 10.1242/dev.02777 [DOI] [PubMed] [Google Scholar]
  74. Kondo RP, Anderson RH, Kupershmidt S, Roden DM, Evans SM. 2003. Development of the cardiac conduction system as delineated by minK-lacZ. J Cardiovasc Electrophysiol 14: 383–391. 10.1046/j.1540-8167.2003.02467.x [DOI] [PubMed] [Google Scholar]
  75. Kreuzberg MM, Söhl G, Kim JS, Verselis VK, Willecke K, Bukauskas FF. 2005. Functional properties of mouse connexin30.2 expressed in the conduction system of the heart. Circ Res 96: 1169–1177. 10.1161/01.RES.0000169271.33675.05 [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. Kreuzberg MM, Schrickel JW, Ghanem A, Kim JS, Degen J, Janssen-Bienhold U, Lewalter T, Tiemann K, Willecke K. 2006. Connexin30.2 containing gap junction channels decelerate impulse propagation through the atrioventricular node. Proc Natl Acad Sci 103: 5959–5964. 10.1073/pnas.0508512103 [DOI] [PMC free article] [PubMed] [Google Scholar]
  77. Lakatta EG, DiFrancesco D. 2009. What keeps us ticking: a funny current, a calcium clock, or both? J Mol Cell Cardiol 47: 157–170. 10.1016/j.yjmcc.2009.03.022 [DOI] [PMC free article] [PubMed] [Google Scholar]
  78. Lee JH, Protze SI, Laksman Z, Backx PH, Keller GM. 2017. Human pluripotent stem cell-derived atrial and ventricular cardiomyocytes develop from distinct mesoderm populations. Cell Stem Cell 21: 179–194.e4. 10.1016/j.stem.2017.07.003 [DOI] [PubMed] [Google Scholar]
  79. Le Scouarnec S, Bhasin N, Vieyres C, Hund TJ, Cunha SR, Koval O, Marionneau C, Chen B, Wu Y, Demolombe S, et al. 2008. Dysfunction in ankyrin-B-dependent ion channel and transporter targeting causes human sinus node disease. Proc Natl Acad Sci 105: 15617–15622. 10.1073/pnas.0805500105 [DOI] [PMC free article] [PubMed] [Google Scholar]
  80. Liang X, Wang G, Lin L, Lowe J, Zhang Q, Bu L, Chen Y, Chen J, Sun Y, Evans SM. 2013. HCN4 dynamically marks the first heart field and conduction system precursors. Circ Res 113: 399–407. 10.1161/CIRCRESAHA.113.301588 [DOI] [PMC free article] [PubMed] [Google Scholar]
  81. Liang X, Zhang Q, Cattaneo P, Zhuang S, Gong X, Spann NJ, Jiang C, Cao X, Zhao X, Zhang X, et al. 2015. Transcription factor ISL1 is essential for pacemaker development and function. J Clin Invest 125: 3256–3268. 10.1172/JCI68257 [DOI] [PMC free article] [PubMed] [Google Scholar]
  82. Liu Y, Bai R, Wang L, Zhang C, Zhao R, Wan D, Chen X, Caceres G, Barr D, Barajas-Martinez H, et al. 2013. Identification of a novel de novo mutation associated with PRKAG2 cardiac syndrome and early onset of heart failure. PLoS ONE 8: e64603 10.1371/journal.pone.0064603 [DOI] [PMC free article] [PubMed] [Google Scholar]
  83. Liu F, Lu MM, Patel NN, Schillinger KJ, Wang T, Patel VV. 2015. GATA-binding factor 6 contributes to atrioventricular node development and function. Circ Cardiovasc Genet 8: 284–293. 10.1161/CIRCGENETICS.113.000587 [DOI] [PMC free article] [PubMed] [Google Scholar]
  84. Lo CW. 2000. Role of gap junctions in cardiac conduction and development: insights from the connexin knockout mice. Circ Res 87: 346–348. 10.1161/01.RES.87.5.346 [DOI] [PubMed] [Google Scholar]
  85. Low SK, Takahashi A, Ebana Y, Ozaki K, Christophersen IE, Ellinor PT, AFGen Consortium, Ogishima S, Yamamoto M, Satoh M, et al. 2017. Identification of six new genetic loci associated with atrial fibrillation in the Japanese population. Nat Genet 49: 953–958. 10.1038/ng.3842 [DOI] [PubMed] [Google Scholar]
  86. Luna-Zurita L, Stirnimann CU, Glatt S, Kaynak BL, Thomas S, Baudin F, Samee MA, He D, Small EM, Mileikovsky M, et al. 2016. Complex interdependence regulates heterotypic transcription factor distribution and coordinates cardiogenesis. Cell 164: 999–1014. 10.1016/j.cell.2016.01.004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  87. Ma L, Lu MF, Schwartz RJ, Martin JF. 2005. Bmp2 is essential for cardiac cushion epithelial–mesenchymal transition and myocardial patterning. Development 132: 5601–5611. 10.1242/dev.02156 [DOI] [PubMed] [Google Scholar]
  88. Mazgalev TN, Tchou PJ. 2000. Surface potentials from the region of the atrioventricular node and their relation to dual pathway electrophysiology. Circulation 101: 2110–2117. 10.1161/01.CIR.101.17.2110 [DOI] [PubMed] [Google Scholar]
  89. Meysen S, Marger L, Hewett KW, Jarry-Guichard T, Agarkova I, Chauvin JP, Perriard JC, Izumo S, Gourdie RG, Mangoni ME, et al. 2007. Nkx2.5 cell-autonomous gene function is required for the postnatal formation of the peripheral ventricular conduction system. Dev Biol 303: 740–753. 10.1016/j.ydbio.2006.12.044 [DOI] [PubMed] [Google Scholar]
  90. Miake J, Marbán E, Nuss HB. 2002. Biological pacemaker created by gene transfer. Nature 419: 132–133. 10.1038/419132b [DOI] [PubMed] [Google Scholar]
  91. Milano A, Vermeer AM, Lodder EM, Barc J, Verkerk AO, Postma AV, van der Bilt IA, Baars MJ, van Haelst PL, Caliskan K, et al. 2014. HCN4 mutations in multiple families with bradycardia and left ventricular noncompaction cardiomyopathy. J Am Coll Cardiol 64: 745–756. 10.1016/j.jacc.2014.05.045 [DOI] [PubMed] [Google Scholar]
  92. Miquerol L, Moreno-Rascon N, Beyer S, Dupays L, Meilhac SM, Buckingham ME, Franco D, Kelly RG. 2010. Biphasic development of the mammalian ventricular conduction system. Circ Res 107: 153–161. 10.1161/CIRCRESAHA.110.218156 [DOI] [PubMed] [Google Scholar]
  93. Mohan RA, Mommersteeg MTM, Domínguez JN, Choquet C, Wakker V, de Gier-de Vries C, Boink GJJ, Boukens BJ, Miquerol L, Verkerk AO, et al. 2018. Embryonic Tbx3+ cardiomyocytes form the mature cardiac conduction system by progressive fate restriction. Development 145: dev167361 10.1242/dev.167361 [DOI] [PubMed] [Google Scholar]
  94. Mohler PJ, Schott JJ, Gramolini AO, Dilly KW, Guatimosim S, duBell WH, Song LS, Haurogné K, Kyndt F, Ali ME, et al. 2003. Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death. Nature 421: 634–639. 10.1038/nature01335 [DOI] [PubMed] [Google Scholar]
  95. Mommersteeg MT, Brown NA, Prall OW, de Gier-de Vries C, Harvey RP, Moorman AF, Christoffels VM. 2007a. Pitx2c and Nkx2-5 are required for the formation and identity of the pulmonary myocardium. Circ Res 101: 902–909. 10.1161/CIRCRESAHA.107.161182 [DOI] [PubMed] [Google Scholar]
  96. Mommersteeg MT, Hoogaars WM, Prall OW, de Gier-de Vries C, Wiese C, Clout DE, Papaioannou VE, Brown NA, Harvey RP, Moorman AF, et al. 2007b. Molecular pathway for the localized formation of the sinoatrial node. Circ Res 100: 354–362. 10.1161/01.RES.0000258019.74591.b3 [DOI] [PubMed] [Google Scholar]
  97. Mommersteeg MT, Domínguez JN, Wiese C, Norden J, de Gier-de Vries C, Burch JB, Kispert A, Brown NA, Moorman AF, Christoffels VM. 2010. The sinus venosus progenitors separate and diversify from the first and second heart fields early in development. Cardiovasc Res 87: 92–101. 10.1093/cvr/cvq033 [DOI] [PubMed] [Google Scholar]
  98. Mori AD, Bruneau BG. 2004. TBX5 mutations and congenital heart disease: Holt-Oram syndrome revealed. Curr Opin Cardiol 19: 211–215. 10.1097/00001573-200405000-00004 [DOI] [PubMed] [Google Scholar]
  99. Mori AD, Zhu Y, Vahora I, Nieman B, Koshiba-Takeuchi K, Davidson L, Pizard A, Seidman JG, Seidman CE, Chen XJ, et al. 2006. Tbx5-dependent rheostatic control of cardiac gene expression and morphogenesis. Dev Biol 297: 566–586. 10.1016/j.ydbio.2006.05.023 [DOI] [PubMed] [Google Scholar]
  100. Moskowitz IP, Pizard A, Patel VV, Bruneau BG, Kim JB, Kupershmidt S, Roden D, Berul CI, Seidman CE, Seidman JG. 2004. The T-Box transcription factor Tbx5 is required for the patterning and maturation of the murine cardiac conduction system. Development 131: 4107–4116. 10.1242/dev.01265 [DOI] [PubMed] [Google Scholar]
  101. Moskowitz IP, Kim JB, Moore ML, Wolf CM, Peterson MA, Shendure J, Nobrega MA, Yokota Y, Berul C, Izumo S, et al. 2007. A molecular pathway including Id2, Tbx5, and Nkx2-5 required for cardiac conduction system development. Cell 129: 1365–1376. 10.1016/j.cell.2007.04.036 [DOI] [PubMed] [Google Scholar]
  102. Munshi NV, McAnally J, Bezprozvannaya S, Berry JM, Richardson JA, Hill JA, Olson EN. 2009. Cx30.2 enhancer analysis identifies Gata4 as a novel regulator of atrioventricular delay. Development 136: 2665–2674. 10.1242/dev.038562 [DOI] [PMC free article] [PubMed] [Google Scholar]
  103. Nguyêñ-Trân VT, Kubalak SW, Minamisawa S, Fiset C, Wollert KC, Brown AB, Ruiz-Lozano P, Barrere-Lemaire S, Kondo R, Norman LW, et al. 2000. A novel genetic pathway for sudden cardiac death via defects in the transition between ventricular and conduction system cell lineages. Cell 102: 671–682. 10.1016/S0092-8674(00)00089-1 [DOI] [PubMed] [Google Scholar]
  104. Pallante BA, Giovannone S, Fang-Yu L, Zhang J, Liu N, Kang G, Dun W, Boyden PA, Fishman GI. 2010. Contactin-2 expression in the cardiac Purkinje fiber network. Circ Arrhythm Electrophysiol 3: 186–194. 10.1161/CIRCEP.109.928820 [DOI] [PMC free article] [PubMed] [Google Scholar]
  105. Pashmforoush M, Lu JT, Chen H, Amand TS, Kondo R, Pradervand S, Evans SM, Clark B, Feramisco JR, Giles W, et al. 2004. Nkx2-5 pathways and congenital heart disease; loss of ventricular myocyte lineage specification leads to progressive cardiomyopathy and complete heart block. Cell 117: 373–386. 10.1016/S0092-8674(04)00405-2 [DOI] [PubMed] [Google Scholar]
  106. Pfeufer A, van Noord C, Marciante KD, Arking DE, Larson MG, Smith AV, Tarasov KV, Müller M, Sotoodehnia N, Sinner MF, et al. 2010. Genome-wide association study of PR interval. Nat Genet 42: 153–159. 10.1038/ng.517 [DOI] [PMC free article] [PubMed] [Google Scholar]
  107. Plotnikov AN, Shlapakova I, Szabolcs MJ, Danilo P Jr, Lorell BH, Potapova IA, Lu Z, Rosen AB, Mathias RT, Brink PR, et al. 2007. Xenografted adult human mesenchymal stem cells provide a platform for sustained biological pacemaker function in canine heart. Circulation 116: 706–713. 10.1161/CIRCULATIONAHA.107.703231 [DOI] [PubMed] [Google Scholar]
  108. Poliak S, Salomon D, Elhanany H, Sabanay H, Kiernan B, Pevny L, Stewart CL, Xu X, Chiu SY, Shrager P, et al. 2003. Juxtaparanodal clustering of Shaker-like K+ channels in myelinated axons depends on Caspr2 and TAG-1. J Cell Biol 162: 1149–1160. 10.1083/jcb.200305018 [DOI] [PMC free article] [PubMed] [Google Scholar]
  109. Potapova I, Plotnikov A, Lu Z, Danilo P, Valiunas V, Qu J, Doronin S, Zuckerman J, Shlapakova IN, Gao J, et al. 2004. Human mesenchymal stem cells as a gene delivery system to create cardiac pacemakers. Circ Res 94: 952–959. 10.1161/01.RES.0000123827.60210.72 [DOI] [PubMed] [Google Scholar]
  110. Protze SI, Liu J, Nussinovitch U, Ohana L, Backx PH, Gepstein L, Keller GM. 2017. Sinoatrial node cardiomyocytes derived from human pluripotent cells function as a biological pacemaker. Nat Biotechnol 35: 56–68. 10.1038/nbt.3745 [DOI] [PubMed] [Google Scholar]
  111. Puskaric S, Schmitteckert S, Mori AD, Glaser A, Schneider KU, Bruneau BG, Blaschke RJ, Steinbeisser H, Rappold G. 2010. Shox2 mediates Tbx5 activity by regulating Bmp4 in the pacemaker region of the developing heart. Hum Mol Genet 19: 4625–4633. 10.1093/hmg/ddq393 [DOI] [PMC free article] [PubMed] [Google Scholar]
  112. Remme CA, Verkerk AO, Hoogaars WM, Aanhaanen WT, Scicluna BP, Annink C, van den Hoff MJ, Wilde AA, van Veen TA, Veldkamp MW, et al. 2009. The cardiac sodium channel displays differential distribution in the conduction system and transmural heterogeneity in the murine ventricular myocardium. Basic Res Cardiol 104: 511–522. 10.1007/s00395-009-0012-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  113. Rentschler S, Vaidya DM, Tamaddon H, Degenhardt K, Sassoon D, Morley GE, Jalife J, Fishman GI. 2001. Visualization and functional characterization of the developing murine cardiac conduction system. Development 128: 1785–1792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  114. Rentschler S, Zander J, Meyers K, France D, Levine R, Porter G, Rivkees SA, Morley GE, Fishman GI. 2002. Neuregulin-1 promotes formation of the murine cardiac conduction system. Proc Natl Acad Sci 99: 10464–10469. 10.1073/pnas.162301699 [DOI] [PMC free article] [PubMed] [Google Scholar]
  115. Rentschler S, Harris BS, Kuznekoff L, Jain R, Manderfield L, Lu MM, Morley GE, Patel VV, Epstein JA. 2011. Notch signaling regulates murine atrioventricular conduction and the formation of accessory pathways. J Clin Invest 121: 525–533. 10.1172/JCI44470 [DOI] [PMC free article] [PubMed] [Google Scholar]
  116. Risebro CA, Petchey LK, Smart N, Gomes J, Clark J, Vieira JM, Yanni J, Dobrzynski H, Davidson S, Zuberi Z, et al. 2012. Epistatic rescue of Nkx2.5 adult cardiac conduction disease phenotypes by prospero-related homeobox protein 1 and HDAC3. Circ Res 111: e19–e31. 10.1161/CIRCRESAHA.111.260695 [DOI] [PubMed] [Google Scholar]
  117. Rubart M, Zipes DP. 2005. Genes and cardiac repolarization: the challenge ahead. Circulation 112: 1242–1244. 10.1161/CIRCULATIONAHA.105.563015 [DOI] [PubMed] [Google Scholar]
  118. Rutenberg JB, Fischer A, Jia H, Gessler M, Zhong TP, Mercola M. 2006. Developmental patterning of the cardiac atrioventricular canal by Notch and Hairy-related transcription factors. Development 133: 4381–4390. 10.1242/dev.02607 [DOI] [PMC free article] [PubMed] [Google Scholar]
  119. Sano M, Kamitsuji S, Kamatani N, Hong KW, Han BG, Kim Y, Kim JW, Aizawa Y, Fukuda K; Japan Pharmacogenomics Data Science Consortium (JPDSC). 2014. Genome-wide association study of electrocardiographic parameters identifies a new association for PR interval and confirms previously reported associations. Hum Mol Genet 23: 6668-6676. 10.1093/hmg/ddu375 [DOI] [PubMed] [Google Scholar]
  120. Shekhar A, Lin X, Liu FY, Zhang J, Mo H, Bastarache L, Denny JC, Cox NJ, Delmar M, Roden DM, et al. 2016. Transcription factor ETV1 is essential for rapid conduction in the heart. J Clin Invest 126: 4444–4459. 10.1172/JCI87968 [DOI] [PMC free article] [PubMed] [Google Scholar]
  121. Singh R, Horsthuis T, Farin HF, Grieskamp T, Norden J, Petry M, Wakker V, Moorman AF, Christoffels VM, Kispert A. 2009. Tbx20 interacts with smads to confine Tbx2 expression to the atrioventricular canal. Circ Res 105: 442–452. 10.1161/CIRCRESAHA.109.196063 [DOI] [PubMed] [Google Scholar]
  122. Singh R, Hoogaars WM, Barnett P, Grieskamp T, Rana MS, Buermans H, Farin HF, Petry M, Heallen T, Martin JF, et al. 2012. Tbx2 and Tbx3 induce atrioventricular myocardial development and endocardial cushion formation. Cell Mol Life Sci 69: 1377–1389. 10.1007/s00018-011-0884-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  123. Smith JG, Magnani JW, Palmer C, Meng YA, Soliman EZ, Musani SK, Kerr KF, Schnabel RB, Lubitz SA, Sotoodehnia N, et al. 2011. Genome-wide association studies of the PR interval in African Americans. PLoS Genet 7: e1001304 10.1371/journal.pgen.1001304 [DOI] [PMC free article] [PubMed] [Google Scholar]
  124. Smits JP, Koopmann TT, Wilders R, Veldkamp MW, Opthof T, Bhuiyan ZA, Mannens MM, Balser JR, Tan HL, Bezzina CR, et al. 2005. A mutation in the human cardiac sodium channel (E161K) contributes to sick sinus syndrome, conduction disease and Brugada syndrome in two families. J Mol Cell Cardiol 38: 969–981. 10.1016/j.yjmcc.2005.02.024 [DOI] [PubMed] [Google Scholar]
  125. Sotoodehnia N, Isaacs A, de Bakker PI, Dörr M, Newton-Cheh C, Nolte IM, van der Harst P, Müller M, Eijgelsheim M, Alonso A, et al. 2010. Common variants in 22 loci are associated with QRS duration and cardiac ventricular conduction. Nat Genet 42: 1068-1076. 10.1038/ng.716 [DOI] [PMC free article] [PubMed] [Google Scholar]
  126. Spach MS, Heidlage JF, Dolber PC, Barr RC. 2000. Electrophysiological effects of remodeling cardiac gap junctions and cell size: experimental and model studies of normal cardiac growth. Circ Res 86: 302–311. 10.1161/01.RES.86.3.302 [DOI] [PubMed] [Google Scholar]
  127. Stanley EG, Biben C, Elefanty A, Barnett L, Koentgen F, Robb L, Harvey RP. 2002. Efficient Cre-mediated deletion in cardiac progenitor cells conferred by a 3′UTR-ires-Cre allele of the homeobox gene Nkx2-5. Int J Dev Biol 46: 431–439. [PubMed] [Google Scholar]
  128. Stefanovic S, Barnett P, van Duijvenboden K, Weber D, Gessler M, Christoffels VM. 2014. GATA-dependent regulatory switches establish atrioventricular canal specificity during heart development. Nat Commun 5: 3680 10.1038/ncomms4680 [DOI] [PMC free article] [PubMed] [Google Scholar]
  129. Stieber J, Herrmann S, Feil S, Loster J, Feil R, Biel M, Hofmann F, Ludwig A. 2003. The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart. Proc Natl Acad Sci 100: 15235–15240. 10.1073/pnas.2434235100 [DOI] [PMC free article] [PubMed] [Google Scholar]
  130. Stroud DM, Gaussin V, Burch JB, Yu C, Mishina Y, Schneider MD, Fishman GI, Morley GE. 2007. Abnormal conduction and morphology in the atrioventricular node of mice with atrioventricular canal targeted deletion of Alk3/Bmpr1a receptor. Circulation 116: 2535–2543. 10.1161/CIRCULATIONAHA.107.696583 [DOI] [PMC free article] [PubMed] [Google Scholar]
  131. Sun Y, Liang X, Najafi N, Cass M, Lin L, Cai CL, Chen J, Evans SM. 2007. Islet 1 is expressed in distinct cardiovascular lineages, including pacemaker and coronary vascular cells. Dev Biol 304: 286–296. 10.1016/j.ydbio.2006.12.048 [DOI] [PMC free article] [PubMed] [Google Scholar]
  132. Sun C, Zhang T, Liu C, Gu S, Chen Y. 2013. Generation of Shox2-Cre allele for tissue specific manipulation of genes in the developing heart, palate, and limb. Genesis 51: 515–522. 10.1002/dvg.22397 [DOI] [PMC free article] [PubMed] [Google Scholar]
  133. Sunyaev S, Ramensky V, Koch I, Lathe W III, Kondrashov AS, Bork P. 2001. Prediction of deleterious human alleles. Hum Mol Genet 10: 591–597. 10.1093/hmg/10.6.591 [DOI] [PubMed] [Google Scholar]
  134. Tessadori F, van Weerd JH, Burkhard SB, Verkerk AO, de Pater E, Boukens BJ, Vink A, Christoffels VM, Bakkers J. 2012. Identification and functional characterization of cardiac pacemaker cells in zebrafish. PLoS ONE 7: e47644 10.1371/journal.pone.0047644 [DOI] [PMC free article] [PubMed] [Google Scholar]
  135. Tse HF, Xue T, Lau CP, Siu CW, Wang K, Zhang QY, Tomaselli GF, Akar FG, Li RA. 2006. Bioartificial sinus node constructed via in vivo gene transfer of an engineered pacemaker HCN channel reduces the dependence on electronic pacemaker in a sick-sinus syndrome model. Circulation 114: 1000–1011. 10.1161/CIRCULATIONAHA.106.615385 [DOI] [PubMed] [Google Scholar]
  136. van den Boogaard M, Wong LY, Tessadori F, Bakker ML, Dreizehnter LK, Wakker V, Bezzina CR, t Hoen PA, Bakkers J, Barnett P, et al. 2012. Genetic variation in T-box binding element functionally affects SCN5A/SCN10A enhancer. J Clin Invest 122: 2519–2530. 10.1172/JCI62613 [DOI] [PMC free article] [PubMed] [Google Scholar]
  137. van den Boogaard M, Smemo S, Burnicka-Turek O, Arnolds DE, van de Werken HJ, Klous P, McKean D, Muehlschlegel JD, Moosmann J, Toka O, et al. 2014. A common genetic variant within SCN10A modulates cardiac SCN5A expression. J Clin Invest 124: 1844–1852. 10.1172/JCI73140 [DOI] [PMC free article] [PubMed] [Google Scholar]
  138. van Eif VWW, Devalla HD, Boink GJJ, Christoffels VM. 2018. Transcriptional regulation of the cardiac conduction system. Nat Rev Cardiol 15: 617–630. 10.1038/s41569-018-0031-y [DOI] [PubMed] [Google Scholar]
  139. Van Mierop LH. 1967. Location of pacemaker in chick embryo heart at the time of initiation of heartbeat. Am J Physiol 212: 407–415. 10.1152/ajplegacy.1967.212.2.407 [DOI] [PubMed] [Google Scholar]
  140. van Setten J, Brody JA, Jamshidi Y, Swenson BR, Butler AM, Campbell H, Del Greco FM, Evans DS, Gibson Q, Gudbjartsson DF, et al. 2018. PR interval genome-wide association meta-analysis identifies 50 loci associated with atrial and atrioventricular electrical activity. Nat Commun 9: 2904 10.1038/s41467-018-04766-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  141. van Setten J, Verweij N, Mbarek H, Niemeijer MN, Trompet S, Arking DE, Brody JA, Gandin I, Grarup N, Hall LM, et al. 2019. Genome-wide association meta-analysis of 30,000 samples identifies seven novel loci for quantitative ECG traits. Eur J Hum Genet 27: 952–962. 10.1038/s41431-018-0295-z [DOI] [PMC free article] [PubMed] [Google Scholar]
  142. Vedantham V, Galang G, Evangelista M, Deo RC, Srivastava D. 2015. RNA sequencing of mouse sinoatrial node reveals an upstream regulatory role for Islet-1 in cardiac pacemaker cells. Circ Res 116: 797–803. 10.1161/CIRCRESAHA.116.305913 [DOI] [PMC free article] [PubMed] [Google Scholar]
  143. Veerman CC, Podliesna S, Tadros R, Lodder EM, Mengarelli I, de Jonge B, Beekman L, Barc J, Wilders R, Wilde AAM, et al. 2017. The Brugada syndrome susceptibility gene HEY2 modulates cardiac transmural ion channel patterning and electrical heterogeneity. Circ Res 121: 537–548. 10.1161/CIRCRESAHA.117.310959 [DOI] [PubMed] [Google Scholar]
  144. Veldkamp MW, Wilders R, Baartscheer A, Zegers JG, Bezzina CR, Wilde AA. 2003. Contribution of sodium channel mutations to bradycardia and sinus node dysfunction in LQT3 families. Circ Res 92: 976–983. 10.1161/01.RES.0000069689.09869.A8 [DOI] [PubMed] [Google Scholar]
  145. Verheijck EE, Wilders R, Joyner RW, Golod DA, Kumar R, Jongsma HJ, Bouman LN, van Ginneken AC. 1998. Pacemaker synchronization of electrically coupled rabbit sinoatrial node cells. J Gen Physiol 111: 95–112. 10.1085/jgp.111.1.95 [DOI] [PMC free article] [PubMed] [Google Scholar]
  146. Vicente-Steijn R, Passier R, Wisse LJ, Schalij MJ, Poelmann RE, Gittenberger-de Groot AC, Jongbloed MR. 2011. Funny current channel HCN4 delineates the developing cardiac conduction system in chicken heart. Heart Rhythm 8: 1254–1263. 10.1016/j.hrthm.2011.03.043 [DOI] [PubMed] [Google Scholar]
  147. Virágh S, Challice CE. 1980. The development of the conduction system in the mouse embryo heart. Dev Biol 80: 28–45. 10.1016/0012-1606(80)90496-0 [DOI] [PubMed] [Google Scholar]
  148. Wang J, Klysik E, Sood S, Johnson RL, Wehrens XH, Martin JF. 2010. Pitx2 prevents susceptibility to atrial arrhythmias by inhibiting left-sided pacemaker specification. Proc Natl Acad Sci 107: 9753–9758. 10.1073/pnas.0912585107 [DOI] [PMC free article] [PubMed] [Google Scholar]
  149. Wang J, Bai Y, Li N, Ye W, Zhang M, Greene SB, Tao Y, Chen Y, Wehrens XH, Martin JF. 2014. Pitx2-microRNA pathway that delimits sinoatrial node development and inhibits predisposition to atrial fibrillation. Proc Natl Acad Sci 111: 9181–9186. 10.1073/pnas.1405411111 [DOI] [PMC free article] [PubMed] [Google Scholar]
  150. Weinberger F, Mehrkens D, Friedrich FW, Stubbendorff M, Hua X, Müller JC, Schrepfer S, Evans SM, Carrier L, Eschenhagen T. 2012. Localization of Islet-1-positive cells in the healthy and infarcted adult murine heart. Circ Res 110: 1303–1310. 10.1161/CIRCRESAHA.111.259630 [DOI] [PMC free article] [PubMed] [Google Scholar]
  151. Wessels A, Vermeulen JL, Verbeek FJ, Virágh S, Kálmán F, Lamers WH, Moorman AF. 1992. Spatial distribution of “tissue-specific” antigens in the developing human heart and skeletal muscle. III: An immunohistochemical analysis of the distribution of the neural tissue antigen G1N2 in the embryonic heart; implications for the development of the atrioventricular conduction system. Anat Rec 232: 97–111. 10.1002/ar.1092320111 [DOI] [PubMed] [Google Scholar]
  152. Wiese C, Grieskamp T, Airik R, Mommersteeg MT, Gardiwal A, de Gier-de Vries C, Schuster-Gossler K, Moorman AF, Kispert A, Christoffels VM. 2009. Formation of the sinus node head and differentiation of sinus node myocardium are independently regulated by Tbx18 and Tbx3. Circ Res 104: 388–397. 10.1161/CIRCRESAHA.108.187062 [DOI] [PubMed] [Google Scholar]
  153. Wu M, Peng S, Zhao Y. 2014a. Inducible gene deletion in the entire cardiac conduction system using Hcn4-CreERT2 BAC transgenic mice. Genesis 52: 134–140. 10.1002/dvg.22730 [DOI] [PMC free article] [PubMed] [Google Scholar]
  154. Wu M, Peng S, Yang J, Tu Z, Cai X, Cai CL, Wang Z, Zhao Y. 2014b. Baf250a orchestrates an epigenetic pathway to repress the Nkx2.5-directed contractile cardiomyocyte program in the sinoatrial node. Cell Res 24: 1201–1213. 10.1038/cr.2014.113 [DOI] [PMC free article] [PubMed] [Google Scholar]
  155. Xin M, Small EM, van Rooij E, Qi X, Richardson JA, Srivastava D, Nakagawa O, Olson EN. 2007. Essential roles of the bHLH transcription factor Hrt2 in repression of atrial gene expression and maintenance of postnatal cardiac function. Proc Natl Acad Sci 104: 7975–7980. 10.1073/pnas.0702447104 [DOI] [PMC free article] [PubMed] [Google Scholar]
  156. Xue T, Cho HC, Akar FG, Tsang SY, Jones SP, Marbán E, Tomaselli GF, Li RA. 2005. Functional integration of electrically active cardiac derivatives from genetically engineered human embryonic stem cells with quiescent recipient ventricular cardiomyocytes: insights into the development of cell-based pacemakers. Circulation 111: 11–20. 10.1161/01.CIR.0000151313.18547.A2 [DOI] [PubMed] [Google Scholar]
  157. Yang L, Soonpaa MH, Adler ED, Roepke TK, Kattman SJ, Kennedy M, Henckaerts E, Bonham K, Abbott GW, Linden RM, et al. 2008. Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature 453: 524–528. 10.1038/nature06894 [DOI] [PubMed] [Google Scholar]
  158. Ye W, Wang J, Song Y, Yu D, Sun C, Liu C, Chen F, Zhang Y, Wang F, Harvey RP, et al. 2015. A common Shox2-Nkx2-5 antagonistic mechanism primes the pacemaker cell fate in the pulmonary vein myocardium and sinoatrial node. Development 142: 2521–2532. 10.1242/dev.120220 [DOI] [PMC free article] [PubMed] [Google Scholar]
  159. Yoo S, Dobrzynski H, Fedorov VV, Xu SZ, Yamanushi TT, Jones SA, Yamamoto M, Nikolski VP, Efimov IR, Boyett MR. 2006. Localization of Na+ channel isoforms at the atrioventricular junction and atrioventricular node in the rat. Circulation 114: 1360–1371. 10.1161/CIRCULATIONAHA.106.613182 [DOI] [PubMed] [Google Scholar]
  160. Zhang SS, Kim KH, Rosen A, Smyth JW, Sakuma R, Delgado-Olguin P, Davis M, Chi NC, Puviindran V, Gaborit N, et al. 2011. Iroquois homeobox gene 3 establishes fast conduction in the cardiac His-Purkinje network. Proc Natl Acad Sci 108: 13576–13581. 10.1073/pnas.1106911108 [DOI] [PMC free article] [PubMed] [Google Scholar]
  161. Zhang JZ, Termglinchan V, Shao NY, Itzhaki I, Liu C, Ma N, Tian L, Wang VY, Chang ACY, Guo H, et al. 2019. A human iPSC double-reporter system enables purification of cardiac lineage subpopulations with distinct function and drug response profiles. Cell Stem Cell 24: 802–811.e5. 10.1016/j.stem.2019.02.015 [DOI] [PMC free article] [PubMed] [Google Scholar]

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