3D models of the cardiac conduction system in healthy neonatal human hearts.

Iatrogenic damage to the cardiac conduction system (CCS) remains a significant risk during congenital heart surgery. Current surgical best practice involves using superficial anatomical landmarks to locate and avoid damaging the CCS. Prior work indicates inherent variability in the anatomy of the CCS and supporting tissues. This study introduces high-resolution, 3D models of the CCS in normal pediatric human hearts to evaluate variability in the nodes and surrounding structures. Human pediatric hearts were obtained with an average donor age of 2.7 days. A pipeline was developed to excise, section, stain, and image atrioventricular (AVN) and sinus nodal (SN) tissue regions. A convolutional neural network was trained to enable precise multi-class segmentation of whole-slide images, which were subsequently used to generate high- resolution 3D tissue models. Nodal tissue region models were created. All models (10 AVN, 8 SN) contain tissue composition of neural tissue, vasculature, and nodal tissues at micrometer resolution. We describe novel nodal anatomical variations. We found that the depth of the His bundle in females was on average 304 µm shallower than those of male patients. These models provide surgeons with insight into the heterogeneity of the nodal regions and the intricate relationships between the CCS and surrounding structures.

Spatially resolved multiomics of human cardiac niches.

The function of a cell is defined by its intrinsic characteristics and its niche: the tissue microenvironment in which it dwells. Here we combine single-cell and spatial transcriptomics data to discover cellular niches within eight regions of the human heart. We map cells to microanatomical locations and integrate knowledge-based and unsupervised structural annotations. We also profile the cells of the human cardiac conduction system1. The results revealed their distinctive repertoire of ion channels, G-protein-coupled receptors (GPCRs) and regulatory networks, and implicated FOXP2 in the pacemaker phenotype. We show that the sinoatrial node is compartmentalized, with a core of pacemaker cells, fibroblasts and glial cells supporting glutamatergic signalling. Using a custom CellPhoneDB.org module, we identify trans-synaptic pacemaker cell interactions with glia. We introduce a druggable target prediction tool, drug2cell, which leverages single-cell profiles and drug-target interactions to provide mechanistic insights into the chronotropic effects of drugs, including GLP-1 analogues. In the epicardium, we show enrichment of both IgG+ and IgA+ plasma cells forming immune niches that may contribute to infection defence. Overall, we provide new clarity to cardiac electro-anatomy and immunology, and our suite of computational approaches can be applied to other tissues and organs.

Anatomical study of the cardiac conduction system in swine hearts.

The cardiac conduction system (CCS) is crucial for regulating heartbeats; therefore, clinicians and comedicals involved in cardiovascular medicine treatment must have a thorough understanding of the CCS structure and function. However, anatomical education of the CCS based on actual dissection and observation is uncommon, although such educational methodology promotes three-dimensional structural understanding of the observed object. Based on previous studies, we examined the CCS structure in the heart of a swine (pig, Sus scrofa domestica) which has been used in the biological, medical and anatomical curricula as science teaching materials, by using macroscopic dissection procedures. Most CCS structures in a young pig heart were successfully identified and illustrated on a macroscopic scale. The atrioventricular bundle (His bundle) was located on the lower edge of the membranous interventricular septum and was clearly distinguished from the general myocardial fibres by its colour and fibre arrangement direction. Following the atrioventricular bundle towards the atrium or ventricle with properly removing the endocardium and myocardium, the atrioventricular node or the right and left bundles appeared respectively. In contrast, the sinoatrial node was not identified. The anatomy of the CCS in young pig hearts was essentially similar to that previously reported in humans and several domestic animals. Our findings of the CCS in young pig hearts are expected to be useful for medical and anatomical education for medical and comedical students, young clinicians and comedical workers.

Anatomy of the conduction tissues 100 years on: what have we learned?

Knowledge of the anatomy of the 'conduction tissues' of the heart is a 20th century phenomenon. Although controversies still continue on the topic, most could have been avoided had greater attention been paid to the original descriptions. All cardiomyocytes, of course, have the capacity to conduct the cardiac impulse. The tissues specifically described as 'conducting' first generate the cardiac impulse, and then deliver it in such a fashion that the ventricles contract in orderly fashion. The tissues cannot readily be distinguished by gross inspection. Robust definitions for their recognition had been provided by the end of the first decade of the 20th century. These definitions retain their currency. The sinus node lies as a cigar-shaped structure subepicardially within the terminal groove. There is evidence that it is associated with a paranodal area that may have functional significance. Suggestions of dual nodes, however, are without histological confirmation. The atrioventricular node is located within the triangle of Koch, with significant inferior extensions occupying the atrial vestibules and with septal connections. The conduction axis penetrates the insulating plane of the atrioventricular junctions to continue as the ventricular pathways. Remnants of a ring of cardiomyocytes observed during development are also to be found within the atrial vestibules, particularly a prominent retroaortic remnant, although that their role has still to be determined. Application of the initial criteria for nodes and tracts shows that there are no special 'conducting tissues' in the pulmonary venous sleeves that might underscore the abnormal rhythm of atrial fibrillation.

Localization of the sinoatrial and atrioventricular nodal region in neonatal and juvenile ovine hearts.

Localization of the components of the cardiac conduction system (CCS) is essential for many therapeutic procedures in cardiac surgery and interventional cardiology. While histological studies provided fundamental insights into CCS localization, this information is incomplete and difficult to translate to aid in intraprocedural localization. To advance our understanding of CCS localization, we set out to establish a framework for quantifying nodal region morphology. Using this framework, we quantitatively analyzed the sinoatrial node (SAN) and atrioventricular node (AVN) in ovine with postmenstrual age ranging from 4.4 to 58.3 months. In particular, we studied the SAN and AVN in relation to the epicardial and endocardial surfaces, respectively. Using anatomical landmarks, we excised the nodes and adjacent tissues, sectioned those at a thickness of 4 µm at 100 µm intervals, and applied Masson's trichrome stain to the sections. These sections were then imaged, segmented to identify nodal tissue, and analyzed to quantify nodal depth and superficial tissue composition. The minimal SAN depth ranged between 20 and 926 µm. AVN minimal depth ranged between 59 and 1192 µm in the AVN extension region, 49 and 980 µm for the compact node, and 148 and 888 µm for the transition to His Bundle region. Using a logarithmic regression model, we found that minimal depth increased logarithmically with age for the AVN (R2 = 0.818, P = 0.002). Also, the myocardial overlay of the AVN was heterogeneous within different regions and decreased with increasing age. Age associated alterations of SAN minimal depth were insignificant. Our study presents examples of characteristic tissue patterns superficial to the AVN and within the SAN. We suggest that the presented framework provides quantitative information for CCS localization. Our studies indicate that procedural methods and localization approaches in regions near the AVN should account for the age of patients in cardiac surgery and interventional cardiology.

A novel method with which to visualize the human sinuatrial node: Application for a better understanding of the gross anatomy of this part of the conduction system.

For various clinical/surgical procedures, it is important to accurately understand the location of the sinuatrial node (SAN). Therefore, this study's goal was to develop a new and simple method to visualize the SAN in human hearts. A total of 16 formalin-fixed human hearts were used in the study. After the epicardium was removed, the fat tissue on the myocardium's surface was brushed and removed in a solution of 40°C water with a surfactant to show the SAN's location. Once the structure considered to be the SAN was observed, histological observation was conducted with Masson's trichrome staining to confirm its identity. The working myocardium, SAN branch of the coronary artery, and the structure believed to be the SAN were observed in all specimens. Histological analysis confirmed this structure to be the SAN. We believe that the method described herein might contribute to a better understanding of the SAN's morphologic features and serve as an improved teaching aide. Clin. Anat. 33:232-236, 2020. © 2019 Wiley Periodicals, Inc.

Histological and morphometric study of the components of the sinus and atrioventricular nodes in horses and dogs.

The cardiac nodes are the source of the electrical impulse that is transmitted to the heart, the aim of this work is study the histological and morphometric characteristics of the different components of the sinus and atrioventricular nodes in horses and dogs that help to know the physiopathology of these nodes. A group of ten horse hearts and five dog hearts were used. The region of the sinus and atrioventricular nodes was sectioned serially, and the block of tissue removed for study. The samples were assessed using a morphometric analysis with the Image-Pro Plus 7.1 software and the acquisition of the images using a Leica DMD108 optic microscope. The shape of the horse's sinus node is oblong and its P cells are large. The shape of the dog's sinus is rounded or oblong. The P cells are large and pale. The area of P cells in horses was 976 (SD 223.7) µm2 and in dogs the area for P cells was 106 (SD 30.4) µm2, which indicates that the value for P cells in horses are significantly higher than in dogs (p = .001). The horse atrioventricular node presented an oblong shape and in dogs, presents a spindle shape. The lower cell density in any of the cardiac nodes, especially in P cells of sinus node, can decrease electrical conduction within the nodes and in the internodal tracts, which would reflect the presence of cardiac arrhythmias derived from poor conduction, even in morphologically normal hearts.

Sinus venosus incorporation: contentious issues and operational criteria for developmental and evolutionary studies.

The sinus venosus is a cardiac chamber upstream of the right atrium that harbours the dominant cardiac pacemaker. During human heart development, the sinus venosus becomes incorporated into the right atrium. However, from the literature it is not possible to deduce the characteristics and importance of this process of incorporation, due to inconsistent terminology and definitions in the description of multiple lines of evidence. We reviewed the literature regarding the incorporation of the sinus venosus and included novel electrophysiological data. Most mammals that have an incorporated sinus venosus show a loss of a functional valve guard of the superior caval vein together with a loss of the electrical sinuatrial delay between the sinus venosus and the right atrium. However, these processes are not necessarily intertwined and in a few species only the sinuatrial delay may be lost. Sinus venosus incorporation can be characterised as the loss of the sinuatrial delay of which the anatomical and molecular underpinnings are not yet understood.

Canine and human sinoatrial node: differences and similarities in the structure, function, molecular profiles, and arrhythmia.

The sinoatrial node (SAN) is the primary pacemaker in canine and human hearts. The SAN in both species has a unique three-dimensional heterogeneous structure characterized by small pacemaker myocytes enmeshed within fibrotic strands, which partially insulate the cells from aberrant atrial activation. The SAN pacemaker tissue expresses a unique signature of proteins and receptors that mediate SAN automaticity, ion channel currents, and cell-to-cell communication, which are predominantly similar in both species. Recent intramural optical mapping, integrated with structural and molecular studies, has revealed the existence of up to five specialized SAN conduction pathways that preferentially conduct electrical activation to atrial tissues. The intrinsic heart rate, intranodal leading pacemaker shifts, and changes in conduction in response to physiological and pathophysiological stimuli are similar. Structural and/or functional impairments due to cardiac diseases including heart failure cause SAN dysfunctions (SNDs) in both species. These dysfunctions are usually manifested as severe bradycardia, tachy-brady arrhythmias, and conduction abnormalities including exit block and SAN reentry, which could lead to atrial tachycardia and fibrillation, cardiac arrest, and heart failure. Pharmaceutical drugs and implantable pacemakers are only partially successful in managing SNDs, emphasizing a critical need to develop targeted mechanism-based therapies to treat SNDs. Because several structural and functional characteristics are similar between the canine and human SAN, research in these species may be mutually beneficial for developing novel treatment approaches. This review describes structural, functional, and molecular similarities and differences between the canine and human SAN, with special emphasis on arrhythmias and unique causal mechanisms of SND in diseased hearts.

Dynamic Cellular Integration Drives Functional Assembly of the Heart's Pacemaker Complex.

Impulses generated by a multicellular, bioelectric signaling center termed the sinoatrial node (SAN) stimulate the rhythmic contraction of the heart. The SAN consists of a network of electrochemically oscillating pacemaker cells encased in a heterogeneous connective tissue microenvironment. Although the cellular composition of the SAN has been a point of interest for more than a century, the biological processes that drive the tissue-level assembly of the cells within the SAN are unknown. Here, we demonstrate that the SAN's structural features result from a developmental process during which mesenchymal cells derived from a multipotent progenitor structure, the proepicardium, integrate with and surround pacemaker myocardium. This process actively remodels the forming SAN and is necessary for sustained electrogenic signal generation and propagation. Collectively, these findings provide experimental evidence for how the microenvironmental architecture of the SAN is patterned and demonstrate that proper cellular arrangement is critical for cardiac pacemaker biorhythmicity.

Variação nos tipos de irrigação do nodo sinoatrial em cães de diferentes raças / Variation in irrigation of the sinoatrial node in dogs of different breeds

Considerando a heterogeneidade anatômica e eletrofisiológica do nodo sinoatrial para a geração e propagação do potencial de ação, bem como as particularidades relacionadas a origem da sua irrigação sanguínea, este trabalho teve como objetivo analisar o comportamento das artérias envolvidas na irrigação do nodo sinoatrial em cães realizando uma análise descritiva e comparativa entre diferentes raças estudadas, detalhando a origem, o percurso e a ramescência dos vasos, assim como a eventual ocorrência de anastomoses. Ao todo analisamos resultados obtidos em 240 corações, os quais foram fixados em solução de formalina 10% e submetidos a diafanização de Spalteholz. A irrigação deste ocorre mediante colaterais oriundos do ramo circunflexo direito ou ramo circunflexo esquerdo, mostrando particularidades diferentes para cada raça. Assim, predominantemente, nas raças ora estudadas a irrigação arterial do nodo sinoatrial depende exclusivamente (63,6%), ou de anastomoses de colaterais da artéria coronária direita, havendo também participação dos ramo proximal atrial direito e intermédio atrial direito. Menos frequentemente (15,4%) o suprimento sanguíneo ocorre exclusivamente por conta do ramo proximal atrial esquerdo, oriundo do ramo circunflexo da artéria coronária esquerda. Os dados aqui apresentados sobre a origem da irrigação sanguínea do nodo sinoatrial e a ramescência dos vasos envolvidos nesta tarefa representam conhecimento fundamental para o desenvolvimento da clínica-cirúrgica em cães, da patologia e trabalhos de natureza experimental.(AU)

Considering the anatomical and electrophysiological heterogeneity of the sinoatrial node for generation and propagation of the action potential, as well as the particularities relating to the origin of blood nutrition, this study aimed to analyze the behavior of arteries involved in irrigation of the sinoatrial node in dogs performing a descriptive and comparative analysis between different breeds, with emphasis in the origin, pathway and branching of vessels, as well as the presence of anastomoses. Totally, 240 hearts were fixed in solution of formalin 10% and subjected to Spalteholz diaphanization. The vascularization of the sinoatrial node occurs by the right circumflex branch or left circumflex branch, showing several particularities according to the breed. Thus, predominantly in the studied breeds, the blood supply of the sinoatrial node depends exclusively (63.6%) or from anastomosis of the right coronary artery. There is also participation of right atrial proximal branch and right atrial intermediary branch. Less often (15.4%) the blood supply occurs exclusively from the left atrial proximal branch, which is a branch of the circumflex branch of the left coronary artery. In summary, our results related to the origin of the sinoatrial node blood nutrition and the branching of vessels involved on that represents a fundamental knowledge for the development and improvement of surgery in dogs, as well as for pathology and experimental research.(AU)

Computational assessment of the functional role of sinoatrial node exit pathways in the human heart.

AIM: The human right atrium and sinoatrial node (SAN) anatomy is complex. Optical mapping experiments suggest that the SAN is functionally insulated from atrial tissue except at discrete SAN-atrial electrical junctions called SAN exit pathways, SEPs. Additionally, histological imaging suggests the presence of a secondary pacemaker close to the SAN. We hypothesise that a) an insulating border-SEP anatomical configuration is related to SAN arrhythmia; and b) a secondary pacemaker, the paranodal area, is an alternate pacemaker but accentuates tachycardia. A 3D electro-anatomical computational model was used to test these hypotheses. METHODS: A detailed 3D human SAN electro-anatomical mathematical model was developed based on our previous anatomical reconstruction. Electrical activity was simulated using tissue specific variants of the Fenton-Karma action potential equations. Simulation experiments were designed to deploy this complex electro-anatomical system to assess the roles of border-SEPs and paranodal area by mimicking experimentally observed SAN arrhythmia. Robust and accurate numerical algorithms were implemented for solving the mono domain reaction-diffusion equation implicitly, calculating 3D filament traces, and computing dominant frequency among other quantitative measurements. RESULTS: A centre to periphery gradient of increasing diffusion was sufficient to permit initiation of pacemaking at the centre of the 3D SAN. Re-entry within the SAN, micro re-entry, was possible by imposing significant SAN fibrosis in the presence of the insulating border. SEPs promoted the micro re-entry to generate more complex SAN-atrial tachycardia. Simulation of macro re-entry, i.e. re-entry around the SAN, was possible by inclusion of atrial fibrosis in the presence of the insulating border. The border shielded the SAN from atrial tachycardia. However, SAN micro-structure intercellular gap junctional coupling and the paranodal area contributed to prolonged atrial fibrillation. Finally, the micro-structure was found to be sufficient to explain shifts of leading pacemaker site location. CONCLUSIONS: The simulations establish a relationship between anatomy and SAN electrical function. Microstructure, in the form of intercellular gap junction coupling, was found to regulate SAN function and arrhythmia.

High resolution 3-Dimensional imaging of the human cardiac conduction system from microanatomy to mathematical modeling.

Cardiac arrhythmias and conduction disturbances are accompanied by structural remodelling of the specialised cardiomyocytes known collectively as the cardiac conduction system. Here, using contrast enhanced micro-computed tomography, we present, in attitudinally appropriate fashion, the first 3-dimensional representations of the cardiac conduction system within the intact human heart. We show that cardiomyocyte orientation can be extracted from these datasets at spatial resolutions approaching the single cell. These data show that commonly accepted anatomical representations are oversimplified. We have incorporated the high-resolution anatomical data into mathematical simulations of cardiac electrical depolarisation. The data presented should have multidisciplinary impact. Since the rate of depolarisation is dictated by cardiac microstructure, and the precise orientation of the cardiomyocytes, our data should improve the fidelity of mathematical models. By showing the precise 3-dimensional relationships between the cardiac conduction system and surrounding structures, we provide new insights relevant to valvar replacement surgery and ablation therapies. We also offer a practical method for investigation of remodelling in disease, and thus, virtual pathology and archiving. Such data presented as 3D images or 3D printed models, will inform discussions between medical teams and their patients, and aid the education of medical and surgical trainees.

Computer simulations of reentrant activity in the rabbit sinoatrial node.

With the aid of detailed computer simulations, we have estimated distributions of membrane potential and ionic currents in the core region of a sinoatrial node reentry. We observe reduced amplitudes of the measured quantities in the core; the core sizes for potential and currents did not always coincide. Simulations revealed that acetylcholine, when applied in the vicinity of unstable reentry, attracted the reentry to become the core and to stabilize its rotation. Anatomically detailed simulations of sinoatrial node and surrounding atrial tissue revealed that reentry always rotated around small strips of connective tissue. Acetylcholine superfusion over superior part of the sinoatrial node resulted in a drift of reentry in the cranial direction. Under the latter conditions, reentry may coexist with the pacemaker in the caudal part of the sinoatrial node. Copyright © 2016 John Wiley & Sons, Ltd.

The Cardiac Conduction System: Generation and Conduction of the Cardiac Impulse.

In this article, the authors outline the key components behind the automated generation of the cardiac impulses and the effect these impulses have on cardiac myocytes. Also, a description of the key components of the normal cardiac conduction system is provided, including the sinoatrial node, the atrioventricular node, the His bundle, the bundle branches, and the Purkinje network. Finally, an outline of how each stage of the cardiac conduction system is represented on the electrocardiogram is described, allowing the reader of the electrocardiogram to translate background information about the normal cardiac conduction system to everyday clinical practice.

Current concepts of anatomy and electrophysiology of the sinus node.

The sinoatrial node, or sinus node, of humans is the principal pacemaker of the heart. Over the last century, studies have unraveled the complex molecular architecture of the sinus node and the expression of unique ion channels within its specialized myocytes. Aim of this review is to describe the embriology, the anatomy, the histology and the electrophisiology of the sinus node.

Anatomical Variations in the Sinoatrial Nodal Artery: A Meta-Analysis and Clinical Considerations.

BACKGROUND AND OBJECTIVE: The sinoatrial nodal artery (SANa) is a highly variable vessel which supplies blood to the sinoatrial node (SAN). Due to its variability and susceptibility to iatrogenic injury, our study aimed to assess the anatomy of the SANa and determine the prevalence of its anatomical variations. STUDY DESIGN: An extensive search of major electronic databases was performed to identify all articles reporting anatomical data on the SANa. No lower date limit or language restrictions were applied. Anatomical data regarding the artery were extracted and pooled into a meta-analysis. RESULTS: Sixty-six studies (n = 21455 hearts) were included in the meta-analysis. The SANa usually arose as a single vessel with a pooled prevalence of 95.5% (95%CI:93.6-96.9). Duplication and triplication of the artery were also observed with pooled prevalence of 4.3% (95%CI:2.8-6.0) and 0.3% (95%CI:0-0.7), respectively. The most common origin of the SANa was from the right coronary artery (RCA), found in 68.0% (95%CI:55.6-68.9) of cases, followed by origin from the left circumflex artery, and origin from the left coronary artery with pooled prevalence of 22.1% (95%CI:15.0-26.2) and 2.7 (95%CI:0.7-5.2), respectively. A retrocaval course of the SANa was the most common course of the artery with a pooled prevalence of 47.1% (95%CI:36.0-55.5). The pooled prevalence of an S-shaped SANa was 7.6% (95%CI:2.9-14.1). CONCLUSIONS: The SANa is most commonly reported as a single vessel, originating from the RCA, and taking a retrocaval course to reach the SAN. Knowledge of high risk anatomical variants of the SANa, such as an S-shaped artery, must be taken into account by surgeons to prevent iatrogenic injuries. Specifically, interventional or cardiosurgical procedures, such as the Cox maze procedure for atrial fibrillation, open heart surgeries through the right atrium or intraoperative cross-clamping or dissection procedures during mitral valve surgery using the septal approach can all potentiate the risk for injury in the setting of high-risk morphological variants of the SANa.

Human sinoatrial node structure: 3D microanatomy of sinoatrial conduction pathways.

INTRODUCTION: Despite a century of extensive study on the human sinoatrial node (SAN), the structure-to-function features of specialized SAN conduction pathways (SACP) are still unknown and debated. We report a new method for direct analysis of the SAN microstructure in optically-mapped human hearts with and without clinical history of SAN dysfunction. METHODS: Two explanted donor human hearts were coronary-perfused and optically-mapped. Structural analyses of histological sections parallel to epicardium (∼13-21 µm intervals) were integrated with optical maps to create 3D computational reconstructions of the SAN complex. High-resolution fiber fields were obtained using 3D Eigen-analysis of the structure tensor, and used to analyze SACP microstructure with a fiber-tracking approach. RESULTS: Optical mapping revealed normal SAN activation of the atria through a lateral SACP proximal to the crista terminalis in Heart #1 but persistent SAN exit block in diseased Heart #2. 3D structural analysis displayed a functionally-observed SAN border composed of fibrosis, fat, and/or discontinuous fibers between SAN and atria, which was only crossed by several branching myofiber tracts in SACP regions. Computational 3D fiber-tracking revealed that myofiber tracts of SACPs created continuous connections between SAN #1 and atria, but in SAN #2, SACP region myofiber tracts were discontinuous due to fibrosis and fat. CONCLUSIONS: We developed a new integrative functional, structural and computational approach that allowed for the resolution of the specialized 3D microstructure of human SACPs for the first time. Application of this integrated approach will shed new light on the role of the specialized SAN microanatomy in maintaining sinus rhythm.

Anatomy and pathology of the sinus node.

PURPOSE: The aim of this study is to review the key features of anatomy of the sinus node of the human sinus node and its pathological variations as reported in the literature and supplemented with our observations. RESULTS: The sinus node is located adjacent to important structures such as the right phrenic nerve and the nodal artery. Although intimately related to its matrix of fibrous tissue, the nodal cells are in contact with atrial myocardium through nodal extensions. Nodal cell numbers decrease with age as part of the normal aging process. Pathological changes include diminished size of node and nodal cell replacement with fibro-fatty tissues. CONCLUSIONS: The location of the normal sinus node is well recognised. There is variation in the relationship of the node and its arterial supply to the terminal crest and the cavo-atrial junction.

Anatomical study on the sinuatrial nodal branch in the human coronary artery.

The sinuatrial nodal branch (SANB) of the coronary artery is anatomically important as it irrigates the sinuatrial node. Past studies on the origin and route of the SANB in the human heart have produced discrepant findings. Therefore, in this study, we macroscopically investigated the origin, route, and distribution of the SANB in 293 human hearts. In addition, we examined the relationship between coronary artery dominance and the origin of the SANB. The SANB was found to have one branch in 267 specimens (91.2%), two branches in 25 specimens (8.5%), and three branches in one (0.3%) specimen. The SANB originated from the right coronary artery in 184 branches (57.5%) and from the left coronary artery (circumflex artery) in 136 branches (42.5%). Nine SANB routes were classified, all of which were distributed in the interatrial septum except for one heart with a type R3 SANB course, which was found to extend through the muscle layer of the interatrial septum into the septum. The composition of these courses could relate to the generation of the proepicardium in the amniote.