Neural cell adhesion molecule is required for ventricular conduction system development.

The most distal portion of the ventricular conduction system (VCS) contains cardiac Purkinje cells (PCs), which are essential for synchronous activation of the ventricular myocardium. Contactin-2 (CNTN2), a member of the immunoglobulin superfamily of cell adhesion molecules (IgSF-CAMs), was previously identified as a marker of the VCS. Through differential transcriptional profiling, we discovered two additional highly enriched IgSF-CAMs in the VCS: NCAM-1 and ALCAM. Immunofluorescence staining showed dynamic expression patterns for each IgSF-CAM during embryonic and early postnatal stages, but ultimately all three proteins became highly enriched in mature PCs. Mice deficient in NCAM-1, but not CNTN2 or ALCAM, exhibited defects in PC gene expression and VCS patterning, as well as cardiac conduction disease. Moreover, using ST8sia2 and ST8sia4 knockout mice, we show that inhibition of post-translational modification of NCAM-1 by polysialic acid leads to disrupted trafficking of sarcolemmal intercalated disc proteins to junctional membranes and abnormal expansion of the extracellular space between apposing PCs. Taken together, our data provide insights into the complex developmental biology of the ventricular conduction system.

Development of the Cardiac Conduction System.

The electrical impulses that coordinate the sequential, rhythmic contractions of the atria and ventricles are initiated and tightly regulated by the specialized tissues of the cardiac conduction system. In the mature heart, these impulses are generated by the pacemaker cardiomyocytes of the sinoatrial node, propagated through the atria to the atrioventricular node where they are delayed and then rapidly propagated to the atrioventricular bundle, right and left bundle branches, and finally, the peripheral ventricular conduction system. Each of these specialized components arise by complex patterning events during embryonic development. This chapter addresses the origins and transcriptional networks and signaling pathways that drive the development and maintain the function of the cardiac conduction system.

Cardiac Development and Animal Models of Congenital Heart Defects.

The major events of cardiac development, including early heart formation, chamber morphogenesis and septation, and conduction system and coronary artery development, are briefly reviewed together with a short introduction to the animal species commonly used to study heart development and model congenital heart defects (CHDs).

GATA6 is a regulator of sinus node development and heart rhythm.

The sinus node (SAN) is the primary pacemaker of the human heart, and abnormalities in its structure or function cause sick sinus syndrome, the most common reason for electronic pacemaker implantation. Here we report that transcription factor GATA6, whose mutations in humans are linked to arrhythmia, is highly expressed in the SAN and its haploinsufficiency in mice results in hypoplastic SANs and rhythm abnormalities. Cell-specific deletion reveals a requirement for GATA6 in various SAN lineages. Mechanistically, GATA6 directly activates key regulators of the SAN genetic program in conduction and nonconduction cells, such as TBX3 and EDN1, respectively. The data identify GATA6 as an important regulator of the SAN and provide a molecular basis for understanding the conduction abnormalities associated with GATA6 mutations in humans. They also suggest that GATA6 may be a potential modifier of the cardiac pacemaker.

The evolving state of cardiac resynchronization therapy and conduction system pacing: 25 years of research at EP Europace journal.

Cardiac resynchronization therapy (CRT) was proposed in the 1990s as a new therapy for patients with heart failure and wide QRS with depressed left ventricular ejection fraction despite optimal medical treatment. This review is aimed first to describe the rationale and the physiologic effects of CRT. The journey of the landmark randomized trials leading to the adoption of CRT in the guidelines since 2005 is also reported showing the high level of evidence for CRT. Different alternative pacing modalities of CRT to conventional left ventricular pacing through the coronary sinus have been proposed to increase the response rate to CRT such as multisite pacing and endocardial pacing. A new emerging alternative technique to conventional biventricular pacing, conduction system pacing (CSP), is a promising therapy. The different modalities of CSP are described (Hirs pacing and left bundle branch area pacing). This new technique has to be evaluated in clinical randomized trials before implementation in the guidelines with a high level of evidence.

Fetal Supraventricular Tachycardia: Histologic Evidence of Accessory Pathways Due to Incomplete Annulus Fibrosus Formation.

BACKGROUND: Atrioventricular (AV) reentrant tachycardia is a common type of supraventricular tachycardia (SVT) that occurs in the fetus and neonate. Although many tachycardias resolve within several weeks of birth or respond to medical management, disruptions in the cardiac annulus fibrosus and development of additional accessory pathways may lead to refractory dysrhythmia resulting in fetal hydrops and ultimately, fetal death. OBJECTIVES: While accessory pathways have been well documented anatomically in adult and childhood tachyarrhythmias, there are no reports of the histology of these pathways in human fetuses with SVT. RESEARCH DESIGN, SUBJECTS, MEASURES: This is a small case series of 2 fetuses with a history of SVT that resulted in fetal hydrops. RESULTS: In both cases, examination of the cardiac conduction system was unremarkable and examination of the atrioventricular junction revealed a focally thinned and/or discontinuous annulus fibrosus with documented direct continuity between the atrial and ventricular myocardium in 1 case. CONCLUSIONS: This case series demonstrates that thinning or absence of the annulus fibrosus is a feature seen in fetal SVT, and the development of subsequent aberrant AV connections due to defective formation of the annulus fibrosus suggests a possible cause for these arrhythmias.

Knockout of the Cardiac Transcription Factor NKX2-5 Results in Stem Cell-Derived Cardiac Cells with Typical Purkinje Cell-like Signal Transduction and Extracellular Matrix Formation.

The human heart controls blood flow, and therewith enables the adequate supply of oxygen and nutrients to the body. The correct function of the heart is coordinated by the interplay of different cardiac cell types. Thereby, one can distinguish between cells of the working myocardium, the pace-making cells in the sinoatrial node (SAN) and the conduction system cells in the AV-node, the His-bundle or the Purkinje fibres. Tissue-engineering approaches aim to generate hiPSC-derived cardiac tissues for disease modelling and therapeutic usage with a significant improvement in the differentiation quality of myocardium and pace-making cells. The differentiation of cells with cardiac conduction system properties is still challenging, and the produced cell mass and quality is poor. Here, we describe the generation of cardiac cells with properties of the cardiac conduction system, called conduction system-like cells (CSLC). As a primary approach, we introduced a CrispR-Cas9-directed knockout of the NKX2-5 gene in hiPSC. NKX2-5-deficient hiPSC showed altered connexin expression patterns characteristic for the cardiac conduction system with strong connexin 40 and connexin 43 expression and suppressed connexin 45 expression. Application of differentiation protocols for ventricular- or SAN-like cells could not reverse this connexin expression pattern, indicating a stable regulation by NKX2-5 on connexin expression. The contraction behaviour of the hiPSC-derived CSLCs was compared to hiPSC-derived ventricular- and SAN-like cells. We found that the contraction speed of CSLCs resembled the expected contraction rate of human conduction system cells. Overall contraction was reduced in differentiated cells derived from NKX2-5 knockout hiPSC. Comparative transcriptomic data suggest a specification of the cardiac subtype of CSLC that is distinctly different from ventricular or pacemaker-like cells with reduced myocardial gene expression and enhanced extracellular matrix formation for improved electrical insulation. In summary, knockout of NKX2-5 in hiPSC leads to enhanced differentiation of cells with cardiac conduction system features, including connexin expression and contraction behaviour.

Myocardial development in crocodylians.

BACKGROUND: Recent reports confirmed the notion that there exists a rudimentary cardiac conduction system (CCS) in the crocodylian heart, and development of its ventricular part is linked to septation. We thus analyzed myocardial development with the emphasis on the CCS components and vascularization in two different crocodylian species. RESULTS: Using optical mapping and HNK-1 immunostaining, pacemaker activity was localized to the right-sided sinus venosus. The atrioventricular conduction was restricted to dorsal part of the atrioventricular canal. Within the ventricle, the impulse was propagated from base-to-apex initially by the trabeculae, later by the ventricular septum, in which strands of HNK-1 positivity were temporarily observed. Completion of ventricular septation correlated with transition of ventricular epicardial activation pattern to mature apex-to-base direction from two periapical foci. Despite a gradual thickening of the ventricular wall, no morphological differentiation of the Purkinje network was observed. Thin-walled coronary vessels with endothelium positive for QH1 obtained a smooth muscle coat after septation. Intramyocardial vessels were abundant especially in the rapidly thickening left ventricular wall. CONCLUSIONS: Most of the CCS components present in the homeiotherm hearts can be identified in the developing crocodylian heart, with a notable exception of the Purkinje network distinct from the trabeculae carneae.

A novel transgenic Cre allele to label mouse cardiac conduction system.

The cardiac conduction system is a network of heterogeneous cell population that initiates and propagates electric excitations in the myocardium. Purkinje fibers, a network of specialized myocardial cells, comprise the distal end of the conduction system in the ventricles. The developmental origins of Purkinje fibers and their roles during cardiac physiology and arrhythmia have been reported. However, it is not clear if they play a role during ischemic injury and heart regeneration. Here we introduce a novel tamoxifen-inducible Cre allele that specifically labels a broad range of components in the cardiac conduction system while excludes other cardiac cell types and vital organs. Using this new allele, we investigated the cellular and molecular response of Purkinje fibers to myocardial injury. In a neonatal mouse myocardial infarction model, we observed significant increase in Purkinje cell number in regenerating myocardium. RNA-Seq analysis using laser-captured Purkinje fibers showed a unique transcriptomic response to myocardial infarction. Our finds suggest a novel role of cardiac Purkinje fibers in heart injury.

Nkx2-5 defines a subpopulation of pacemaker cells and is essential for the physiological function of the sinoatrial node in mice.

The sinoatrial node (SAN), the primary cardiac pacemaker, consists of a head domain and a junction/tail domain that exhibit different functional properties. However, the underlying molecular mechanism defining these two pacemaker domains remains elusive. Nkx2-5 is a key transcription factor essential for the formation of the working myocardium, but it was generally thought to be detrimental to SAN development. However, Nkx2-5 is expressed in the developing SAN junction, suggesting a role for Nkx2-5 in SAN junction development and function. In this study, we present unambiguous evidence that SAN junction cells exhibit unique action potential configurations intermediate to those manifested by the SAN head and the surrounding atrial cells, suggesting a specific role for the junction cells in impulse generation and in SAN-atrial exit conduction. Single-cell RNA-seq analyses support this concept. Although Nkx2-5 inactivation in the SAN junction did not cause a malformed SAN at birth, the mutant mice manifested sinus node dysfunction. Thus, Nkx2-5 defines a population of pacemaker cells in the transitional zone. Despite Nkx2-5 being dispensable for SAN morphogenesis during embryogenesis, its deletion hampers atrial activation by the pacemaker.

Computed tomography imaging-identified location and electrocardiographic characteristics of left bundle branch area pacing in bradycardia patients.

INTRODUCTION: Left bundle branch area pacing (LBBAP) is a novel physiological pacing modality. The relationship between the pacing lead tip location and paced electrocardiographic (ECG) characteristics remains unclear. The objectives are to determine the lead tip location within the interventricular septum (IVS) and assess the location-based ECG QRS duration (QRSd) and left ventricular activation time (LVAT). METHODS: This multicenter study enrolled 50 consecutive bradycardia patients who met pacemaker therapy guidelines and received LBBAP implantation via the trans-ventricular septal approach. After at least 3 months postimplant, 12-lead ECGs and pacing parameters were obtained. Cardiac computed tomography (CT) imaging was performed to assess the LBBAP lead tip distance from the LV blood pool. RESULTS: Among the 50 patients, analyzable CT images were obtained in 42. In 23 of the 42 patients, the lead tips were within 2 mm to the LV blood pool (the LV subendocardial (LVSE) group), 13 between 2 and 4 mm (the Near-LVSE group), and the remaining 6 beyond 4 mm (the Mid-LV septal (Mid-LVS) group). No significant differences in paced QRSd were found among the three groups (LVSE, 107 ± 15 ms; Near-LVSE, 106 ± 13 ms; Mid-LVS, 104 ± 15 ms; p = .87). LVAT in the LVSE (64 ± 7 ms) was significantly shorter than in the Mid-LVS (72 ± 8 ms; p < .05), but not significantly different from that in the Near-LVSE (69 ± 8 ms; p > .05). CONCLUSION: In routine LBBAP practice, paced narrow QRSd and fast LVAT, indicative of physiological pacing, were consistently achieved for lead tip location in the LV subendocardial or near LV subendocardial region.

Anatomy of the heart with the highest heart rate.

Shrews occupy the lower extreme of the seven orders of magnitude mammals range in size. Their hearts are large relative to body weight and heart rate can exceed a thousand beats a minute. It is not known whether traits typical of mammal hearts scale to these extremes. We assessed the heart of three species of shrew (genus Sorex) following the sequential segmental analysis developed for human hearts. Using micro-computed tomography, we describe the overall structure and find, in agreement with previous studies, a large and elongate ventricle. The atrial and ventricular septums and the atrioventricular (AV) and arterial valves are typically mammalian. The ventricular walls comprise mostly compact myocardium and especially the right ventricle has few trabeculations on the luminal side. A developmental process of compaction is thought to reduce trabeculations in mammals, but in embryonic shrews the volume of trabeculations increase for every gestational stage, only slower than the compact volume. By expression of Hcn4, we identify a sinus node and an AV conduction axis which is continuous with the ventricular septal crest. Outstanding traits include pulmonary venous sleeve myocardium that reaches farther into the lungs than in any other mammals. Typical proportions of coronary arteries-to-aorta do not scale and the shrew coronary arteries are proportionally enormous, presumably to avoid the high resistance to blood flow of narrow vessels. In conclusion, most cardiac traits do scale to the miniscule shrews. The shrew heart, nevertheless, stands out by its relative size, elongation, proportionally large coronary vessels, and extent of pulmonary venous myocardium.

Transcriptional Patterning of the Ventricular Cardiac Conduction System.

RATIONALE: The heartbeat is organized by the cardiac conduction system (CCS), a specialized network of cardiomyocytes. Patterning of the CCS into atrial node versus ventricular conduction system (VCS) components with distinct physiology is essential for the normal heartbeat. Distinct node versus VCS physiology has been recognized for more than a century, but the molecular basis of this regional patterning is not well understood. OBJECTIVE: To study the genetic and genomic mechanisms underlying node versus VCS distinction and investigate rhythm consequences of failed VCS patterning. METHODS AND RESULTS: Using mouse genetics, we found that the balance between T-box transcriptional activator, Tbx5, and T-box transcriptional repressor, Tbx3, determined the molecular and functional output of VCS myocytes. Adult VCS-specific removal of Tbx5 or overexpression of Tbx3 re-patterned the fast VCS into slow, nodal-like cells based on molecular and functional criteria. In these cases, gene expression profiling showed diminished expression of genes required for VCS-specific fast conduction but maintenance of expression of genes required for nodal slow conduction physiology. Action potentials of Tbx5-deficient VCS myocytes adopted nodal-specific characteristics, including increased action potential duration and cellular automaticity. Removal of Tbx5 in vivo precipitated inappropriate depolarizations in the atrioventricular (His)-bundle associated with lethal ventricular arrhythmias. TBX5 bound and directly activated cis-regulatory elements at fast conduction channel genes required for fast physiological characteristics of the VCS action potential, defining the identity of the adult VCS. CONCLUSIONS: The CCS is patterned entirely as a slow, nodal ground state, with a T-box dependent, physiologically dominant, fast conduction network driven specifically in the VCS. Disruption of the fast VCS gene regulatory network allowed nodal physiology to emerge, providing a plausible molecular mechanism for some lethal ventricular arrhythmias.

Transcriptome profile of the sinoatrial ring reveals conserved and novel genetic programs of the zebrafish pacemaker.

BACKGROUND: Sinoatrial Node (SAN) is part of the cardiac conduction system, which controls the rhythmic contraction of the vertebrate heart. The SAN consists of a specialized pacemaker cell population that has the potential to generate electrical impulses. Although the SAN pacemaker has been extensively studied in mammalian and teleost models, including the zebrafish, their molecular nature remains inadequately comprehended. RESULTS: To characterize the molecular profile of the zebrafish sinoatrial ring (SAR) and elucidate the mechanism of pacemaker function, we utilized the transgenic line sqet33mi59BEt to isolate cells of the SAR of developing zebrafish embryos and profiled their transcriptome. Our analyses identified novel candidate genes and well-known conserved signaling pathways involved in pacemaker development. We show that, compared to the rest of the heart, the zebrafish SAR overexpresses several mammalian SAN pacemaker signature genes, which include hcn4 as well as those encoding calcium- and potassium-gated channels. Moreover, genes encoding components of the BMP and Wnt signaling pathways, as well as members of the Tbx family, which have previously been implicated in pacemaker development, were also overexpressed in the SAR. Among SAR-overexpressed genes, 24 had human homologues implicated in 104 different ClinVar phenotype entries related to various forms of congenital heart diseases, which suggest the relevance of our transcriptomics resource to studying human heart conditions. Finally, functional analyses of three SAR-overexpressed genes, pard6a, prom2, and atp1a1a.2, uncovered their novel role in heart development and physiology. CONCLUSION: Our results established conserved aspects between zebrafish and mammalian pacemaker function and revealed novel factors implicated in maintaining cardiac rhythm. The transcriptome data generated in this study represents a unique and valuable resource for the study of pacemaker function and associated heart diseases.

Embryonic Tbx3+ cardiomyocytes form the mature cardiac conduction system by progressive fate restriction.

A small network of spontaneously active Tbx3+ cardiomyocytes forms the cardiac conduction system (CCS) in adults. Understanding the origin and mechanism of development of the CCS network are important steps towards disease modeling and the development of biological pacemakers to treat arrhythmias. We found that Tbx3 expression in the embryonic mouse heart is associated with automaticity. Genetic inducible fate mapping revealed that Tbx3+ cells in the early heart tube are fated to form the definitive CCS components, except the Purkinje fiber network. At mid-fetal stages, contribution of Tbx3+ cells was restricted to the definitive CCS. We identified a Tbx3+ population in the outflow tract of the early heart tube that formed the atrioventricular bundle. Whereas Tbx3+ cardiomyocytes also contributed to the adjacent Gja5+ atrial and ventricular chamber myocardium, embryonic Gja5+ chamber cardiomyocytes did not contribute to the Tbx3+ sinus node or to atrioventricular ring bundles. In conclusion, the CCS is established by progressive fate restriction of a Tbx3+ cell population in the early developing heart, which implicates Tbx3 as a useful tool for developing strategies to study and treat CCS diseases.

Three-dimensional visualization of the bovine cardiac conduction system and surrounding structures compared to the arrangements in the human heart.

In the human heart, the atrioventricular node is located toward the apex of the triangle of Koch, which is also at the apex of the inferior pyramidal space. It is adjacent to the atrioventricular portion of the membranous septum, through which it penetrates to become the atrioventricular bundle. Subsequent to its penetration, the conduction axis is located on the crest of the ventricular septum, sandwiched between the muscular septum and ventricular component of the membranous septum, where it gives rise to the ramifications of the left bundle branch. In contrast, the bovine conduction axis has a long non-branching component, which penetrates into a thick muscular atrioventricular septum having skirted the main cardiac bone and the rightward half of the non-coronary sinus of the aortic root. It commonly gives rise to both right and left bundle branches within the muscular ventricular septum. Unlike the situation in man, the left bundle branch is long and thin before it branches into its fascicles. These differences from the human heart, however, have yet to be shown in three-dimensions relative to the surrounding structures. We have now achieved this goal by injecting contrast material into the insulating sheaths that surround the conduction network, evaluating the results by subsequent computed tomography. The fibrous atrioventricular membranous septum of the human heart is replaced in the ox by the main cardiac bone and the muscular atrioventricular septum. The apex of the inferior pyramidal space, which in the bovine, as in the human, is related to the atrioventricular node, is placed inferiorly relative to the left ventricular outflow tract. The bovine atrioventricular conduction axis, therefore, originates from a node itself located inferiorly compared to the human arrangement. The axis must then skirt the non-coronary sinus of the aortic root prior to penetrating the thicker muscular ventricular septum, thus accounting for its long non-branching course. We envisage that our findings will further enhance comparative anatomical research.

Transcriptomic Profiling of the Developing Cardiac Conduction System at Single-Cell Resolution.

RATIONALE: The cardiac conduction system (CCS) consists of distinct components including the sinoatrial node, atrioventricular node, His bundle, bundle branches, and Purkinje fibers. Despite an essential role for the CCS in heart development and function, the CCS has remained challenging to interrogate because of inherent obstacles including small cell numbers, large cell-type heterogeneity, complex anatomy, and difficulty in isolation. Single-cell RNA-sequencing allows for genome-wide analysis of gene expression at single-cell resolution. OBJECTIVE: Assess the transcriptional landscape of the entire CCS at single-cell resolution by single-cell RNA-sequencing within the developing mouse heart. METHODS AND RESULTS: Wild-type, embryonic day 16.5 mouse hearts (n=6 per zone) were harvested and 3 zones of microdissection were isolated, including: Zone I-sinoatrial node region; Zone II-atrioventricular node/His region; and Zone III-bundle branch/Purkinje fiber region. Tissue was digested into single-cell suspensions, cells isolated, mRNA reverse transcribed, and barcoded before high-throughput sequencing and bioinformatics analyses. Single-cell RNA-sequencing was performed on over 22 000 cells, and all major cell types of the murine heart were successfully captured including bona fide clusters of cells consistent with each major component of the CCS. Unsupervised weighted gene coexpression network analysis led to the discovery of a host of novel CCS genes, a subset of which were validated using fluorescent in situ hybridization as well as whole-mount immunolabeling with volume imaging (iDISCO+) in 3 dimensions on intact mouse hearts. Further, subcluster analysis unveiled isolation of distinct CCS cell subtypes, including the clinically relevant but poorly characterized transitional cells that bridge the CCS and surrounding myocardium. CONCLUSIONS: Our study represents the first comprehensive assessment of the transcriptional profiles from the entire CCS at single-cell resolution and provides a characterization in the context of development and disease.

Anatomy of the cardiac conduction system.

The specialized cardiomyocytes that constitute the conduction system in the human heart, initiate the electric impulse and result in rhythmic and synchronized contraction of the atria and ventricles. Although the atrioventricular (AV) conduction axis was described more than a century ago by Sunao Tawara, the anatomic pathway for propagation of impulse from atria to the ventricles has been a topic of debate for years. Over the past 2 decades, there has been a resurgence of conduction system pacing (CSP) by implanting pacing leads in the His bundle region in lieu of chronic right ventricular pacing that is associated with worse clinical outcomes. The inherent limitations of implanting the leads in the His bundle region has led to the emergence of left bundle branch area pacing in the past 3 years as an alternative strategy for CSP. The clinical experience from performing CSP has helped electrophysiologists gain deeper insight into the anatomy and physiology of cardiac conduction system. This review details the anatomy of the cardiac conduction system, and highlights some of the recently published articles that aid in better understanding of the AV conduction axis and its variations, the knowledge of which is critical for CSP. The remarkable evolution in technology has led to visualization of the cardiac conduction system using noninvasive, nondestructive high-resolution contrast-enhanced micro-computed tomography imaging that may aid in future CSP. We also discuss from anatomical perspective, the differences seen clinically with His bundle pacing and left bundle branch area pacing.

3D magnetization transfer (MT) for the visualization of cardiac free-running Purkinje fibers: an ex vivo proof of concept.

OBJECTIVES: We investigate the possibility to exploit high-field MRI to acquire 3D images of Purkinje network which plays a crucial role in cardiac function. Since Purkinje fibers (PF) have a distinct cellular structure and are surrounded by connective tissue, we investigated conventional contrast mechanisms along with the magnetization transfer (MT) imaging technique to improve image contrast between ventricular structures of differing macromolecular content. METHODS: Three fixed porcine ventricular samples were used with free-running PFs on the endocardium. T1, T2*, T2, and M0 were evaluated on 2D slices for each sample at 9.4 T. MT parameters were optimized using hard pulses with different amplitudes, offset frequencies and durations. The cardiac structure was assessed through 2D and 3D T1w images with isotropic resolutions of 150 µm. Histology, immunofluorescence, and qPCR were performed to analyze collagen contents of cardiac tissue and PF. RESULTS: An MT preparation module of 350 ms duration inserted into the sequence with a B1 = 10 µT and frequency offset = 3000 Hz showed the best contrast, approximately 0.4 between PFs and myocardium. Magnetization transfer ratio (MTR) appeared higher in the cardiac tissue (MTR = 44.7 ± 3.5%) than in the PFs (MTR = 25.2 ± 6.3%). DISCUSSION: MT significantly improves contrast between PFs and ventricular myocardium and appears promising for imaging the 3D architecture of the Purkinje network.

Molecular Profiling of the Cardiac Conduction System: the Dawn of a New Era.

PURPOSE OF REVIEW: Recent technological advances have led to an increased ability to define the gene expression profile of the cardiac conduction system (CCS). Here, we review the most salient studies to emerge in recent years and discuss existing gaps in our knowledge as well as future areas of investigation. RECENT FINDINGS: Molecular profiling of the CCS spans several decades. However, the advent of high-throughput sequencing strategies has allowed for the discovery of unique transcriptional programs of the many diverse CCS cell types. The CCS, a diverse structure with significant inter- and intra-component cellular heterogeneity, is essential to the normal function of the heart. Progress in transcriptomic profiling has improved the resolution and depth of characterization of these unique and clinically relevant CCS cell types. Future studies leveraging this big data will play a crucial role in improving our understanding of CCS development and function as well as translating these findings into tangible translational tools for the improved detection, prevention, and treatment of cardiac arrhythmias.