Gap Junctional Communication via Connexin43 between Purkinje Fibers and Working Myocytes Explains the Epicardial Activation Pattern in the Postnatal Mouse Left Ventricle.

The mammalian ventricular myocardium forms a functional syncytium due to flow of electrical current mediated in part by gap junctions localized within intercalated disks. The connexin (Cx) subunit of gap junctions have direct and indirect roles in conduction of electrical impulse from the cardiac pacemaker via the cardiac conduction system (CCS) to working myocytes. Cx43 is the dominant isoform in these channels. We have studied the distribution of Cx43 junctions between the CCS and working myocytes in a transgenic mouse model, which had the His-Purkinje portion of the CCS labeled with green fluorescence protein. The highest number of such connections was found in a region about one-third of ventricular length above the apex, and it correlated with the peak proportion of Purkinje fibers (PFs) to the ventricular myocardium. At this location, on the septal surface of the left ventricle, the insulated left bundle branch split into the uninsulated network of PFs that continued to the free wall anteriorly and posteriorly. The second peak of PF abundance was present in the ventricular apex. Epicardial activation maps correspondingly placed the site of the first activation in the apical region, while some hearts presented more highly located breakthrough sites. Taken together, these results increase our understanding of the physiological pattern of ventricular activation and its morphological underpinning through detailed CCS anatomy and distribution of its gap junctional coupling to the working myocardium.

Muscarinic agonists inhibit the ATP-dependent potassium current and suppress the ventricle-Purkinje action potential dispersion.

Activation of the parasympathetic nervous system has been reported to have an antiarrhythmic role during ischemia-reperfusion injury by decreasing the arrhythmia triggers. Furthermore, it was reported that the parasympathetic neurotransmitter acetylcholine is able to modulate the ATP-dependent potassium current (I K-ATP), a crucial current activated during hypoxia. However, the possible significance of this current modulation in the antiarrhythmic mechanism is not fully clarified. Action potentials were measured using the conventional microelectrode technique from canine left ventricular papillary muscle and free-running Purkinje fibers, under normal and hypoxic conditions. Ionic currents were measured using the whole-cell configuration of the patch-clamp method. Acetylcholine at 5 µmol/L did not influence the action potential duration (APD) either in Purkinje fibers or in papillary muscle preparations. In contrast, it significantly lengthened the APD and suppressed the Purkinje-ventricle APD dispersion when it was administered after 5 µmol/L pinacidil application. Carbachol at 3 µmol/L reduced the pinacidil-activated I K-ATP under voltage-clamp conditions. Acetylcholine lengthened the ventricular action potential under simulated ischemia condition. In this study, we found that acetylcholine inhibits the I K-ATP and thus suppresses the ventricle-Purkinje APD dispersion. We conclude that parasympathetic tone may reduce the arrhythmogenic substrate exerting a complex antiarrhythmic mechanism during hypoxic conditions.

3D Bioprinting the Cardiac Purkinje System Using Human Adipogenic Mesenchymal Stem Cell Derived Purkinje Cells.

PURPOSE: The objective of this study was to reprogram human adipogenic mesenchymal stem cells (hADMSCs) to form Purkinje cells and to use the reprogrammed Purkinje cells to bioprint Purkinje networks. METHODS: hADMSCs were reprogrammed to form Purkinje cells using a multi-step process using transcription factors ETS2 and MESP1 to first form cardiac progenitor stem cells followed by SHOX2 and TBX3 to form Purkinje cells. A novel bioprinting method was developed based on Pluronic acid as the sacrificial material and type I collagen as the structural material. The reprogrammed Purkinje cells were used in conjunction with the novel bioprinting method to bioprint Purkinje networks. Printed constructs were evaluated for retention of functional protein connexin 40 (Cx40) and ability to undergo membrane potential changes in response to physiologic stimulus. RESULTS: hADMSCs were successfully reprogrammed to form Purkinje cells based on the expression pattern of IRX3, IRX5, SEMA and SCN10. Reprogrammed purkinje cells were incorporated into a collagen type-1 bioink and the left ventricular Purkinje network was printed using anatomical images of the bovine Purkinje system as reference. Optimization studies demonstrated that 1.8 mg/mL type-I collagen at a seeding density of 300,000 cells per 200 µL resulted in the most functional bioprinted Purkinje networks. Furthermore, bioprinted Purkinje networks formed continuous syncytium, retained expression of vital functional gap junction protein Cx40 post-print, and exhibited membrane potential changes in response to electric stimulation and acetylcholine evaluated by DiBAC4(5), an electrically responsive dye. CONCLUSION: Based on the results of this study, hADMSCs were successfully reprogrammed to form Purkinje cells and bioprinted to form Purkinje networks.

Morphological study of the atrioventricular conduction system and Purkinje fibers in yak.

We studied the morphology of the atrioventricular conduction system (AVCS) and Purkinje fibers of the yak. Light and transmission electron microscopy were used to study the histological features of AVCS. The distributional characteristics of the His-bundle, the left bundle branch (LBB), right bundle branch (RBB), and Purkinje fiber network of yak hearts were examined using gross dissection, ink injection, and ABS casting. The results showed that the atrioventricular node (AVN) of yak located in the right side of interatrial septum and had a flattened ovoid shape. The AVN of yak is composed of the slender, interweaving cells formed almost entirely of the transitional cells (T-cells). The His-bundle extended from the AVN, and split into left LBB and RBB at the crest of the interventricular septum. The LBB descended along the left side of interventricular septum. At approximately the upper 1/3 of the interventricular septum, the LBB typically divided into three branches. The RBB ran under the endocardium of the right side of interventricular septum, and extended to the base of septal papillary muscle, passed into the moderator band, crossed the right ventricular cavity to reach the base of anterior papillary muscle, and divided into four fascicles under the subendocardial layer. The Purkinje fibers in the ventricle formed a complex spatial network. The distributional and cellular component characteristics of the AVCS and Purkinje fibers ensured normal cardiac function.

A quantitative structural and morphometric analysis of the Purkinje network and the Purkinje-myocardial junctions in pig hearts.

The morpho-functional properties of the distal section of the cardiac Purkinje network (PN) and the Purkinje-myocardial junctions (PMJs) are fundamental to understanding the sequence of electrical activation in the heart. The overall structure of the system has already been described, and several computational models have been developed to gain insight into its involvement in cardiac arrhythmias or its interaction with implantable devices, such as pacemakers. However, anatomical descriptions of the PN in the literature have not enabled enough improvements in the accuracy of anatomical-based electrophysiological simulations of the PN in 3D hearts models. In this work, we study the global distribution and morphological properties of the PN, with special emphasis on the cellular and architectural characterization of its intramural branching structure, mesh-like sub-endocardial network, and the PMJs in adult pig hearts by both histopathological and morphometric evaluation. We have defined three main patterns of PMJ: contact through cell bodies, contact through cell prolongations either thick or piliform, and contact through transitional cells. Moreover, from hundreds of micrographs, we quantified the density of PMJs and provided data for the basal/medial/apical regions, anterior/posterior/septal/lateral regions and myocardial/sub-endocardial distribution. Morphometric variables, such as Purkinje cell density and thickness of the bundles, were also analyzed. After combining the results of these parameters, a different septoanterior distribution in the Purkinje cell density was observed towards the cardiac apex, which is associated with a progressive thinning of the conduction bundles and the posterolateral ascension of intramyocardial terminal scattered fibers. The study of the PMJs revealed a decreasing trend towards the base that may anatomically explain the early apical activation. The anterolateral region contains the greatest number of contacts, followed by the anterior and septal regions. This supports the hypothesis that thin distal Purkinje bundles create a junction-rich network that may be responsible for the quick apical depolarization. The PN then ascends laterally and spreads through the anterior and medial walls up to the base. We have established the first morphometric study of the Purkinje system, and provided quantitative and objective data that facilitate its incorporation into the development of models beyond gross and variable pathological descriptions, and which, after further studies, could be useful in the characterization of pathological processes or therapeutic procedures.

Spiral-wave dynamics in a mathematical model of human ventricular tissue with myocytes and Purkinje fibers.

We present systematic numerical studies of the possible effects of the coupling of human endocardial and Purkinje cells at cellular and two-dimensional tissue levels. We find that the autorhythmic-activity frequency of the Purkinje cell in a composite decreases with an increase in the coupling strength; this can even eliminate the autorhythmicity. We observe a delay between the beginning of the action potentials of endocardial and Purkinje cells in a composite; such a delay increases as we decrease the diffusive coupling, and eventually a failure of transmission occurs. An increase in the diffusive coupling decreases the slope of the action-potential-duration-restitution curve of an endocardial cell in a composite. By using a minimal model for the Purkinje network, in which we have a two-dimensional, bilayer tissue, with a layer of Purkinje cells on top of a layer of endocardial cells, we can stabilize spiral-wave turbulence; however, for a sparse distribution of Purkinje-ventricular junctions, at which these two layers are coupled, we can also obtain additional focal activity and many complex transient regimes. We also present additional effects resulting from the coupling of Purkinje and endocardial layers and discuss the relation of our results to the studies performed in anatomically accurate models of the Purkinje network.

Generating Purkinje networks in the human heart.

The Purkinje network is an integral part of the excitation system in the human heart. Yet, to date, there is no in vivo imaging technique to accurately reconstruct its geometry and structure. Computational modeling of the Purkinje network is increasingly recognized as an alternative strategy to visualize, simulate, and understand the role of the Purkinje system. However, most computational models either have to be generated manually, or fail to smoothly cover the irregular surfaces inside the left and right ventricles. Here we present a new algorithm to reliably create robust Purkinje networks within the human heart. We made the source code of this algorithm freely available online. Using Monte Carlo simulations, we demonstrate that the fractal tree algorithm with our new projection method generates denser and more compact Purkinje networks than previous approaches on irregular surfaces. Under similar conditions, our algorithm generates a network with 1219±61 branches, three times more than a conventional algorithm with 419±107 branches. With a coverage of 11±3mm, the surface density of our new Purkije network is twice as dense as the conventional network with 22±7mm. To demonstrate the importance of a dense Purkinje network in cardiac electrophysiology, we simulated three cases of excitation: with our new Purkinje network, with left-sided Purkinje network, and without Purkinje network. Simulations with our new Purkinje network predicted more realistic activation sequences and activation times than simulations without. Six-lead electrocardiograms of the three case studies agreed with the clinical electrocardiograms under physiological conditions, under pathological conditions of right bundle branch block, and under pathological conditions of trifascicular block. Taken together, our results underpin the importance of the Purkinje network in realistic human heart simulations. Human heart modeling has the potential to support the design of personalized strategies for single- or bi-ventricular pacing, radiofrequency ablation, and cardiac defibrillation with the common goal to restore a normal heart rhythm.

Modeling our understanding of the His-Purkinje system.

The His-Purkinje System (HPS) is responsible for the rapid electric conduction in the ventricles. It relays electrical impulses from the atrioventricular node to the muscle cells and, thus, coordinates the contraction of ventricles in order to ensure proper cardiac pump function. The HPS has been implicated in the genesis of ventricular tachycardia and fibrillation as a source of ectopic beats, as well as forming distinct portions of reentry circuitry. Despite its importance, it remains much less well characterized, structurally and functionally, than the myocardium. Notably, important differences exist with regard to cell structure and electrophysiology, including ion channels, intracellular calcium handling, and gap junctions. Very few computational models address the HPS, and the majority of organ level modeling studies omit it. This review will provide an overview of our current knowledge of structure and function (including electrophysiology) of the HPS. We will review the most recent advances in modeling of the system from the single cell to the organ level, with considerations for relevant interspecies distinctions.

TRPM4 non-selective cation channels influence action potentials in rabbit Purkinje fibres.

KEY POINTS: The transient receptor potential melastatin 4 (TRPM4) inhibitor 9-phenanthrol reduces action potential duration in rabbit Purkinje fibres but not in ventricle. TRPM4-like single channel activity is observed in isolated rabbit Purkinje cells but not in ventricular cells. The TRPM4-like current develops during the notch and early repolarization phases of the action potential in Purkinje cells. ABSTRACT: Transient receptor potential melastatin 4 (TRPM4) Ca(2+)-activated non-selective cation channel activity has been recorded in cardiomyocytes and sinus node cells from mammals. In addition, TRPM4 gene mutations are associated with human diseases of cardiac conduction, suggesting that TRPM4 plays a role in this aspect of cardiac function. Here we evaluate the TRPM4 contribution to cardiac electrophysiology of Purkinje fibres. Ventricular strips with Purkinje fibres were isolated from rabbit hearts. Intracellular microelectrodes recorded Purkinje fibre activity and the TRPM4 inhibitor 9-phenanthrol was applied to unmask potential TRPM4 contributions to the action potential. 9-Phenanthrol reduced action potential duration measured at the point of 50 and 90% repolarization with an EC50 of 32.8 and 36.1×10(-6) mol l(-1), respectively, but did not modulate ventricular action potentials. Inside-out patch-clamp recordings were used to monitor TRPM4 activity in isolated Purkinje cells. TRPM4-like single channel activity (conductance = 23.8 pS; equal permeability for Na(+) and K(+); sensitivity to voltage, Ca(2+) and 9-phenanthrol) was observed in 43% of patches from Purkinje cells but not from ventricular cells (0/16). Action potential clamp experiments performed in the whole-cell configuration revealed a transient inward 9-phenanthrol-sensitive current (peak density = -0.65 ± 0.15 pA pF(-1); n = 5) during the plateau phases of the Purkinje fibre action potential. These results show that TRPM4 influences action potential characteristics in rabbit Purkinje fibres and thus could modulate cardiac conduction and be involved in triggering arrhythmias.

Isolation and characterization of embryonic stem cell-derived cardiac Purkinje cells.

The cardiac Purkinje fiber network is composed of highly specialized cardiomyocytes responsible for the synchronous excitation and contraction of the ventricles. Computational modeling, experimental animal studies, and intracardiac electrical recordings from patients with heritable and acquired forms of heart disease suggest that Purkinje cells (PCs) may also serve as critical triggers of life-threatening arrhythmias. Nonetheless, owing to the difficulty in isolating and studying this rare population of cells, the precise role of PC in arrhythmogenesis and the underlying molecular mechanisms responsible for their proarrhythmic behavior are not fully characterized. Conceptually, a stem cell-based model system might facilitate studies of PC-dependent arrhythmia mechanisms and serve as a platform to test novel therapeutics. Here, we describe the generation of murine embryonic stem cells (ESC) harboring pan-cardiomyocyte and PC-specific reporter genes. We demonstrate that the dual reporter gene strategy may be used to identify and isolate the rare ESC-derived PC (ESC-PC) from a mixed population of cardiogenic cells. ESC-PC display transcriptional signatures and functional properties, including action potentials, intracellular calcium cycling, and chronotropic behavior comparable to endogenous PC. Our results suggest that stem-cell derived PC are a feasible new platform for studies of developmental biology, disease pathogenesis, and screening for novel antiarrhythmic therapies.

Towards computational modelling of the human foetal electrocardiogram: normal sinus rhythm and congenital heart block.

AIMS: We aim to engineer a computational model of propagation during normal sinus rhythm in the foetal human heart, by modifying models for adult cardiac tissue to match foetal electrocardiogram (fECG) characteristics. The model will be partially validated by fECG data, and applied to explore possible mechanisms of arrhythmogenesis in the foetal heart. METHODS AND RESULTS: Foetal electrocardiograms have been recorded during pregnancy, with P- and T-waves, and the QRS complex, identified by averaging and signal processing. Intervals of the fECG are extracted and used to modify currently available human adult cardiomyocyte models. RR intervals inform models of the pacemaking cells by constraining their rate, the QT interval and its rate dependence constrain models of ventricular cells, and the width of the P-wave, the QR and PR intervals constrain propagation times, conduction velocities, and intercellular coupling. These cell models are coupled into a one-dimensional (1D) model of propagation during normal sinus rhythm in the human foetal heart. We constructed a modular, heterogeneous 1D model for propagation in the foetal heart, and predicted the effects of reduction in L-type Ca(++) current. These include bradycardia and atrioventricular conduction blocks. These may account quantitatively for congenital heart block produced by positive IgG antibodies. CONCLUSION: The fECG can be interpreted mechanistically and quantitatively by using a simple computational model for propagation. After further validation, by clinical recordings of the fECG and the electrophysiological experiments on foetal cardiac cells and tissues, the model may be used to predict the effects of maternally administered pharmaceuticals on the fECG.

Why do we have Purkinje fibers deep in our heart?

Purkinje fibers were the first discovered component of the cardiac conduction system. Originally described in sheep in 1839 as pale subendocardial cells, they were found to be present, although with different morphology, in all mammalian and avian hearts. Here we review differences in their appearance and extent in different species, summarize the current state of knowledge of their function, and provide an update on markers for these cells. Special emphasis is given to popular model species and human anatomy.

Myocardial Notch signaling reprograms cardiomyocytes to a conduction-like phenotype.

BACKGROUND: Notch signaling has previously been shown to play an essential role in regulating cell fate decisions and differentiation during cardiogenesis in many systems including Drosophila, Xenopus, and mammals. We hypothesized that Notch may also be involved in directing the progressive lineage restriction of cardiomyocytes into specialized conduction cells. METHODS AND RESULTS: In hearts where Notch signaling is activated within the myocardium from early development onward, Notch promotes a conduction-like phenotype based on ectopic expression of conduction system-specific genes and cell autonomous changes in electrophysiology. With the use of an in vitro assay to activate Notch in newborn cardiomyocytes, we observed global changes in the transcriptome, and in action potential characteristics, consistent with reprogramming to a conduction-like phenotype. CONCLUSIONS: Notch can instruct the differentiation of chamber cardiac progenitors into specialized conduction-like cells. Plasticity remains in late-stage cardiomyocytes, which has potential implications for engineering of specialized cardiovascular tissues.

Native presynaptic metabotropic glutamate receptor 4 (mGluR4) interacts with exocytosis proteins in rat cerebellum.

The eight pre- or/and post-synaptic metabotropic glutamatergic receptors (mGluRs) modulate rapid excitatory transmission sustained by ionotropic receptors. They are classified in three families according to their percentage of sequence identity and their pharmacological properties. mGluR4 belongs to group III and is mainly localized presynaptically. Activation of group III mGluRs leads to depression of excitatory transmission, a process that is exclusively provided by mGluR4 at parallel fiber-Purkinje cell synapse in rodent cerebellum. This function relies at least partly on an inhibition of presynaptic calcium influx, which controls glutamate release. To improve the understanding of molecular mechanisms of the mGluR4 depressant effect, we decided to identify the proteins interacting with this receptor. Immunoprecipitations using anti-mGluR4 antibodies were performed with cerebellar extracts. 183 putative partners that co-immunoprecipitated with anti-mGluR4 antibodies were identified and classified according to their cellular functions. It appears that native mGluR4 interacts with several exocytosis proteins such as Munc18-1, synapsins, and syntaxin. In addition, native mGluR4 was retained on a Sepharose column covalently grafted with recombinant Munc18-1, and immunohistochemistry experiments showed that Munc18-1 and mGluR4 colocalized at plasma membrane in HEK293 cells, observations in favor of an interaction between the two proteins. Finally, affinity chromatography experiments using peptides corresponding to the cytoplasmic domains of mGluR4 confirmed the interaction observed between mGluR4 and a selection of exocytosis proteins, including Munc18-1. These results could give indications to explain how mGluR4 can modulate glutamate release at parallel fiber-Purkinje cell synapses in the cerebellum in addition to the inhibition of presynaptic calcium influx.

Inter-model consistency and complementarity: learning from ex-vivo imaging and electrophysiological data towards an integrated understanding of cardiac physiology.

Computational models of the heart at various scales and levels of complexity have been independently developed, parameterised and validated using a wide range of experimental data for over four decades. However, despite remarkable progress, the lack of coordinated efforts to compare and combine these computational models has limited their impact on the numerous open questions in cardiac physiology. To address this issue, a comprehensive dataset has previously been made available to the community that contains the cardiac anatomy and fibre orientations from magnetic resonance imaging as well as epicardial transmembrane potentials from optical mapping measured on a perfused ex-vivo porcine heart. This data was used to develop and customize four models of cardiac electrophysiology with different level of details, including a personalized fast conduction Purkinje system, a maximum a posteriori estimation of the 3D distribution of transmembrane potential, the personalization of a simplified reaction-diffusion model, and a detailed biophysical model with generic conduction parameters. This study proposes the integration of these four models into a single modelling and simulation pipeline, after analyzing their common features and discrepancies. The proposed integrated pipeline demonstrates an increase prediction power of depolarization isochrones in different pacing conditions.

Ionic mechanisms of electrophysiological properties and repolarization abnormalities in rabbit Purkinje fibers.

Purkinje cells play an important role in drug-induced arrhythmogenesis and are widely used in preclinical drug safety assessments. Repolarization abnormalities such as action potential (AP) prolongation and early afterdeploarizations (EAD) are often observed in vitro upon pharmacological interventions. However, because drugs do not act on only one defined target, it is often difficult to fully explain the mechanisms of action and their potential arrhythmogenicity. Computational models, when appropriately detailed and validated, can be used to gain mechanistic insights into the mechanisms of action of certain drugs. Nevertheless, no model of Purkinje electrophysiology that is able to reproduce characteristic Purkinje responses to drug-induced changes in ionic current conductances such as AP prolongation and EAD generation currently exists. In this study, a novel biophysically detailed model of rabbit Purkinje electrophysiology was developed by integration of data from voltage-clamp and AP experimental recordings. Upon validation, we demonstrate that the model reproduces many key electrophysiological properties of rabbit Purkinje cells. These include: AP morphology and duration, both input resistance and rate dependence properties as well as response to hyperkalemia. Pharmacological interventions such as inward rectifier K(+) current and rapid delayed rectifier K(+) current block as well as late Na(+) current increase result in significant AP changes. However, enhanced L-type Ca(2+) current (i(CaL)) dominates in EAD genesis in Purkinje fibers. In addition, i(CaL) inactivation dynamics and intercellular coupling in tissue strongly modulate EAD formation. We conclude that EAD generation in Purkinje cells is mediated by an increase in i(CaL) and modulated by its inactivation kinetics.

Quantified proarrhythmic potential of selected human embryonic stem cell-derived cardiomyocytes.

To improve proarrhythmic predictability of preclinical models, we assessed whether human ventricular-like embryonic stem cell-derived cardiomyocytes (hESC-CMs) can be selected following a standardized protocol. Also, we quantified their arrhythmogenic response and compared this to a contemporary used rabbit Purkinje fiber (PF) model. Multiple transmembrane action potentials (AP) were recorded from 164 hESC-CM clusters (9 different batches), and 12 isolated PFs from New Zealand White rabbits. AP duration (APD), early afterdepolarizations (EADs), triangulation (T), and short-term variability of repolarization (STV) were determined on application of the I(Kr) blocker E-4031 (0.03/0.1/0.3/1 muM). Isoproterenol (0.1 muM) was used to assess adrenergic response. To validate the phenotype, RNA isolated from atrial- and ventricular-like clusters (n=8) was analyzed using low-density Taqman arrays. Based on initial experiments, slow beating rate (<50 bpm) and long APD (>200 ms) were used to select 31 ventricular-like clusters. E-4031 (1 muM) prolonged APD (31/31) and induced EADs only in clusters with APD90>300 ms (11/16). EADs were associated with increased T (1.6+/-0.2 vs 2.0+/-0.3) and STV (2.7+/-1.5 vs 6.9+/-1.9). Rabbit PF reacted in a similar way with regards to EADs (5/12), increased T (1.3+/-0.1 vs 1.9+/-0.4), and STV (1.2+/-0.9 vs 7.1+/-5.6). According to ROC values, hESC-CMs (STV 0.91) could predict EADs at least equivalent to PF (STV 0.69). Isoproterenol shortened APD and completely suppressed EADs. Gene expression analysis revealed that HCN1/2, KCNA5, and GJA5 were higher in atrial/nodal-like cells, whereas KCNJ2 and SCN1B were higher in ventricular-like cells (P<0.05). Selection of hESC-CM clusters with a ventricular-like phenotype can be standardized. The proarrhythmic results are qualitatively and quantitatively comparable between hESC-CMs and rabbit PF. Our results indicate that additional validation of this new safety pharmacology model is warranted.

Contactin-2 expression in the cardiac Purkinje fiber network.

BACKGROUND: Purkinje cells (PCs) comprise the most distal component of the cardiac conduction system, and their unique electrophysiological properties and the anatomic complexity of the Purkinje fiber network may account for the prominent role these cells play in the genesis of various arrhythmic syndromes. METHODS AND RESULTS: Differential transcriptional profiling of murine Purkinje fibers and working ventricular myocytes was performed to identify novel genes expressed in PCs. The most highly enriched transcript in Purkinje fibers encoded Contactin-2 (Cntn2), a cell adhesion molecule critical for neuronal patterning and ion channel clustering. Endogenous expression of Cntn2 in the murine ventricle was restricted to a subendocardial network of myocytes that also express beta-galactosidase in CCS-lacZ transgenic mice and the connexin40 gap junction protein. Both Cntn2-lacZ knockin mice and Cntn2-EGFP BAC transgenic reporter mice confirmed expression of Cntn2 in the Purkinje fiber network, as did immunohistochemical staining of single canine Purkinje fibers. Whole-cell patch-clamp recordings and measurements of Ca(2+) transients in Cntn2-EGFP(+) cells revealed electrophysiological properties indicative of PCs and distinctive from those of cardiac myocytes, including prolonged action potentials and frequent afterdepolarizations. CONCLUSIONS: Cntn2 is a novel marker of the specialized cardiac conduction system. Endogenous expression of Cntn2 as well as Cntn2-dependent transcriptional reporters provides a new tool through which Purkinje cell biology and pathophysiology can now more readily be deciphered. Expression of a contactin family member within the CCS may provide a mechanistic basis for patterning of the conduction system network and the organization of ion channels within Purkinje cells.

Cardiac Purkinje cells.

Purkinje cells are specialized for rapid propagation in the heart. Furthermore, Purkinje fibers as the source as well as the perpetuator of arrhythmias is a familiar finding. This is not surprising considering their location in the heart and their unique cell ultrastructure, cell electrophysiology, and mode of excitation-contraction coupling. This review touches on each of these points as we outline what is known today about Purkinje fibers/cells.

Intramural Purkinje cell network of sheep ventricles as the terminal pathway of conduction system.

To identify the anatomical basis for cardiac electrical signal conduction, particularly seeking the intramural terminals of conduction pathway within the ventricles, sheep hearts were examined compared with human hearts utilizing the characteristic morphology of Purkinje cells as a histological marker. In 15 sheep and five human autopsies of noncardiac death, prevalence of Purkinje or Purkinje-type cells were histologically examined in the atrioventricular node, its distal conduction pathway, the interventricular septum, and the right- and left-ventricular free walls. Myocardial tissue cleavages were examined in the transmural sections (along cardiac base-to-apex axis) obtained from the septum and ventricular free walls. Serial histological sections through virtually the entirety of the septum in selected sheep were used as the basis of a three-dimensional reconstruction of the conduction pathway, particularly of the intramural Purkinje cell network. Purkinje cells were found within the mural myocardium of sheep ventricles whereas no intramural Purkinje-type cell was detected within the human ventricles. In the sheep septum, every intramural Purkinje cell composed a three-dimensional network throughout the mural myocardium, which proximally connected to the subendocardial extension of the bundle branches and distally formed an occasional junction with ordinary working myocytes. The Purkinje-cell network may participate in the ventricular excitation as the terminal conduction pathway. Individual connections among the Purkinje cells contain the links of through-wall orientation which would benefit the signal conduction crossing the architectural barriers by cleavages in sheep hearts. The myocardial architectural changes found in diseased hearts could disrupt the network links including those with transmural orientation. Anat Rec, 2009. (c) 2008 Wiley-Liss, Inc.