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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 system 1 . 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., (© 2023. The Author(s).)

Histologically, the cardiac conduction network is formed of electrically isolated subendocardial fibers that comprise specialized cells with fewer myofibrils and mitochondria than cardiomyocytes. Our aim is to uncover regional variations of cardiac conduction fibers through histological and morphometric study in a porcine and human model. We analyzed five male adult human hearts and five male pig hearts. The left ventricles were dissected and sectioned in the axial plane into three parts: basal, middle third and apex regions. Cardiac conduction fibers study was carried out using hematoxylin-eosin and Masson's trichrome staining, and cardiac conduction cells and their junctions were identified using desmin, and a PAS method. Cardiac conduction fibers were difficult to pinpoint in humans, mostly showing a darker color or equal to cardiomyocytes. Cardiac conduction fibers in humans were in the subendocardium and in pigs in the myocardium and subendocardium. Cardiac conduction fibers were located mainly in the septal region in both humans and pigs. In our morphometric analysis, we were able to determine that cardiac conduction cells in humans (18.52 +/- 5.41 μm) and pigs (21.32 +/- 6.45 μm) were large, compared to cardiomyocytes. Conduction fiber-myocardial junctions were present in 10% in humans and 24.2% in pigs. The performance of immunohistochemical methods made it possible to improve the identification of cardiac conduction cells in the species studied. Study of cardiac conduction fibers and cells and their myocardial junctions is vital to gain insight into their normal distribution in the species analyzed, and thus advance the use of pigs in experimental models of the cardiac conduction system in humans., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) resemble fetal cardiomyocytes and electrical stimulation (ES) has been explored to mature the differentiated cells. Here, we hypothesize that ES applied at the beginning of the differentiation process, triggers both differentiation of the hiPSC-CMs into a specialized conduction system (CS) phenotype and cell maturation. We applied ES for 15 days starting on day 0 of the differentiation process and found an increased expression of transcription factors and proteins associated with the development and function of CS including Irx3, Nkx2.5 and contactin 2, Hcn4 and Scn5a, respectively. We also found activation of intercalated disc proteins (Nrap and β-catenin). We detected ES-induced CM maturation as indicated by increased Tnni1 and Tnni3 expression. Confocal micrographs showed a shift towards expression of the gap junction protein connexin 40 in ES hiPSC-CM compared to the more dominant expression of connexin 43 in controls. Finally, analysis of functional parameters revealed that ES hiPSC-CMs exhibited faster action potential (AP) depolarization, longer intracellular Ca 2+ transients, and slower AP duration at 90% of repolarization, resembling fast conducting fibers. Altogether, we provided evidence that ES during the differentiation of hiPSC to cardiomyocytes lead to development of cardiac conduction-like cells with more mature cytoarchitecture. Thus, hiPSC-CMs exposed to ES during differentiation can be instrumental to develop CS cells for cardiac disease modelling, screening individual drugs on a precison medicine type platform and support the development of novel therapeutics for arrhythmias., Competing Interests: Declaration of competing interest The authors confirm that there are no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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.

Biological pacemakers that combine cell‑based and gene‑based therapies are a promising treatment for sick sinus syndrome or severe atrioventricular block. The current study aimed to induce differentiation of adipose tissue‑derived stem cells (ADSCs) into cardiac pacemaker cells through co‑expression of the transcription factors insulin gene enhancer binding protein 1 (ISL‑1) and T‑box18 (Tbx18). ADSCs were transfected with green fluorescent protein, ISL‑1, Tbx18 or ISL‑1+Tbx18 fluorescent protein lentiviral vectors, and subsequently co‑cultured with neonatal rat ventricular cardiomyocytes in vitro for 7 days. The potential for regulating the differentiation of ADSCs into pacemaker‑like cells was evaluated by cell morphology, beating rate, reverse transcription‑quantitative polymerase chain reaction, western blotting, immunofluorescence and electrophysiological activity. ADSCs were successfully transformed into spontaneously beating cells that exhibited a behavior similar to that of co‑cultured pacemaker cells. This effect was significantly increased in the combined ISL‑1 and Tbx18 group. These results provide a potential strategy for enriching the cardiac pacemaker cell population from ADSCs.

Hybrid approaches combining gene‑ and cell‑based therapies to make biological pacemakers are a promising therapeutic avenue for bradyarrhythmia. The present study aimed to direct adipose tissue‑derived stem cells (ADSCs) to differentiate specifically into cardiac pacemaker cells by overexpressing a single transcription factor, insulin gene enhancer binding protein 1 (ISL‑1). In the present study, the ADSCs were transfected with ISL‑1 or mCherry fluorescent protein lentiviral vectors and co‑cultured with neonatal rat ventricular cardiomyocytes (NRVMs) in vitro for 5‑7 days. The feasibility of regulating the differentiation of ADSCs into pacemaker‑like cells by overexpressing ISL‑1 was evaluated by observation of cell morphology and beating rate, reverse transcription‑quantitative polymerase chain reaction analysis, western blotting, immunofluorescence and analysis of electrophysiological activity. In conclusion, these data indicated that the overexpression of ISL‑1 in ADSCs may enhance the pacemaker phenotype and automaticity in vitro, features which were significantly increased following co‑culture induction.

Two hypotheses have been proposed to explain propagation of the action potential in heart. According to the gap junction hypothesis local short-circuit currents pass from the proximal depolarized cell to the distal inactive cell via gap junctions and are responsible for the depolarization of the distal cell. In the ephapse hypothesis the depolarization of the proximal cell generates an electrical field in the narrow cleft between cells resulting in depolarization beyond threshold of the distal cell. Measurements of length constant, free diffusion of 42 K, local currents between cells, existence of high-conductance gap junctions led to the conclusion that heart muscle is a functional syncytium. Propagation of the action potential, however, is not uniform but anisotropic and discontinuous; it can be also unidirectional. These findings are strong arguments in favor of the gap junction thesis. They do not exclude, as predicted by theoretical calculations, that in conditions of an abnormal fall in gap junction conductance ephaptic conduction takes over. In this last case, definitive experimental confirmation is still required. See also: https://doi.org/10.14814/phy2.13861 & https://doi.org/10.14814/phy2.13862., (© 2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Distinct electrophysiological phenotypes are exhibited by biological cells that have differentiated into particular cell types. The usual approach when simulating the cardiac electrophysiology of tissue that includes different cell types is to model the different cell types as occupying spatially distinct yet coupled regions. Instead, we model the electrophysiology of well-mixed cells by using homogenisation to derive an extension to the commonly used monodomain or bidomain equations. These new equations permit spatial variations in the distribution of the different subtypes of cells and will reduce the computational demands of solving the governing equations. We validate the homogenisation computationally, and then use the new model to explain some experimental observations from stem cell-derived cardiomyocyte monolayers.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are emerging tools for applications such as drug discovery and screening for pro-arrhythmogenicity and cardiotoxicity as leading causes for drug attrition. Understanding the electrophysiology (EP) of hPSC-CMs is essential but conventional manual patch-clamping is highly laborious and low-throughput. Here we adapted hPSC-CMs derived from two human embryonic stem cell (hESC) lines, HES2 and H7, for a 16-channel automated planar-recording approach for single-cell EP characterization. Automated current- and voltage-clamping, with an overall success rate of 55.0 ± 11.3%, indicated that 90% of hPSC-CMs displayed ventricular-like action potential (AP) and the ventricular cardiomyocytes (VCMs) derived from the two hESC lines expressed similar levels of I Na , I CaL , I kr and I f and similarly lacked I to and I K1 . These well-characterized hPSC-VCMs could also be readily adapted for automated assays of pro-arrhythmic drug screening. As an example, we showed that flecainide (FLE) induced I Na blockade, leftward steady-state inactivation shift, slowed recovery from inactivation in our hPSC-VCMs. Since single-cell EP assay is insufficient to predict drug-induced reentrant arrhythmias, hPSC-VCMs were further reassembled into 2D human ventricular cardiac monolayers (hvCMLs) for multi-cellular electrophysiological assessments. Indeed, FLE significantly slowed the conduction velocity while causing AP prolongation. Our RNA-seq data suggested that cell-cell interaction enhanced the maturity of hPSC-VCMs. Taken collectively, a combinatorial approach using single-cell EP and hvCMLs is needed to comprehensively assess drug-induced arrhythmogenicity., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Heart failure (HF) is a clinical syndrome characterized by ventricular contractile dysfunction. About 50% of death in patients with HF are due to fetal ventricular arrhythmias including ventricular tachycardia and ventricular fibrillation. Understanding ventricular arrhythmic substrates and discovering effective antiarrhythmic interventions are extremely important for improving the prognosis of patients with HF and reducing its mortality. In this review, we discussed ventricular arrhythmic substrates and current clinical therapeutics for ventricular arrhythmias in HF. Base on the fact that classic antiarrhythmic drugs have the limited efficacy, side effects, and proarrhythmic potentials, we also updated some therapeutic strategies for the development of potential new antiarrhythmic interventions for patients with HF., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The components of the cardiac conduction system (CCS) generate and propagate the electrical impulse that initiates cardiac contraction. These interconnected components share properties, such as automaticity, that set them apart from the working myocardium of the atria and ventricles. A variety of tools and approaches have been used to define the CCS lineages. These include genetic labeling of cells expressing lineage markers and fate mapping of dye labeled cells, which we will discuss in this review. We conclude that there is not a single CCS lineage, but instead early cell fate decisions segregate the lineages of the CCS components while they remain interconnected. The latter is relevant for development of therapies for conduction system disease that focus on reprogramming cardiomyocytes or instruction of pluripotent stem cells.

Long-range chromosomal interactions bring distal regulatory elements and promoters together to regulate gene expression in biological processes. By performing promoter capture Hi-C (PCHi-C) on human embryonic stem cell-derived cardiomyocytes (hESC-CMs), we show that such promoter interactions are a key mechanism by which enhancers contact their target genes after hESC-CM differentiation from hESCs. We also show that the promoter interactome of hESC-CMs is associated with expression quantitative trait loci (eQTLs) in cardiac left ventricular tissue; captures the dynamic process of genome reorganisation after hESC-CM differentiation; overlaps genome-wide association study (GWAS) regions associated with heart rate; and identifies new candidate genes in such regions. These findings indicate that regulatory elements in hESC-CMs identified by our approach control gene expression involved in ventricular conduction and rhythm of the heart. The study of promoter interactions in other hESC-derived cell types may be of utility in functional investigation of GWAS-associated regions.

Objective: A flexible, efficient, and verifiable pacemaker cell model is essential to the design of real-time virtual hearts that can be used for closed-loop validation of cardiac devices. A new parametric model of pacemaker action potential is developed to address this need., Methods: The action potential phases are modeled using hybrid automaton with one piecewise-linear continuous variable. The model can capture rate-dependent dynamics, such as action potential duration restitution, conduction velocity restitution, and overdrive suppression by incorporating nonlinear update functions. Simulated dynamics of the model compared well with previous models and clinical data., Conclusion: The results show that the parametric model can reproduce the electrophysiological dynamics of a variety of pacemaker cells, such as sinoatrial node, atrioventricular node, and the His-Purkinje system, under varying cardiac conditions., Significance: This is an important contribution toward closed-loop validation of cardiac devices using real-time heart models.

A cell-sourced biological pacemaker is a promising therapeutic approach for sick sinus syndrome (SSS) or severe atrial ventricular block (AVB). Adipose tissue-derived stem cells (ATSCs), which are optimal candidate cells for possible use in regenerative therapy for acute or chronic myocardial injury, have the potential to differentiate into spontaneous beating cardiomyocytes. However, the pacemaker characteristics of the beating cells need to be confirmed, and little is known about the underlying differential mechanism. In this study, we found that brown adipose tissue-derived stem cells (BATSCs) in mice could differentiate into spontaneous beating cells in 15% FBS Dulbecco's modified Eagle's medium (DMEM) without additional treatment. Subsequently, we provide additional evidence, including data regarding ultrastructure, protein expression, electrophysiology, and pharmacology, to support the differentiation of BATSCs into a cardiac pacemaker phenotype during the course of early cultivation. Furthermore, we found that silencing Tbx18, a key transcription factor in the development of pacemaker cells, terminated the differentiation of BATSCs into a pacemaker phenotype, suggesting that Tbx18 is required to direct BATSCs toward a cardiac pacemaker fate. The expression of Tbx3 and shox2, the other two important transcription factors in the development of pacemaker cells, was decreased by silencing Tbx18, which suggests that Tbx18 mediates the differentiation of BATSCs into a pacemaker phenotype via these two downstream transcription factors.

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.

Temperature-induced changes in cardiac output (Q̇) in fish are largely dependent on thermal modulation of heart rate (f H ), and at high temperatures Q̇ collapses due to heat-dependent depression of f H This study tests the hypothesis that firing rate of sinoatrial pacemaker cells sets the upper thermal limit of f H in vivo. To this end, temperature dependence of action potential (AP) frequency of enzymatically isolated pacemaker cells (pacemaker rate, f PM ), spontaneous beating rate of isolated sinoatrial preparations (f SA ), and in vivo f H of the cold-acclimated (4°C) brown trout (Salmo trutta fario) were compared under acute thermal challenges. With rising temperature, f PM steadily increased because of the acceleration of diastolic depolarization and shortening of AP duration up to the break point temperature (T BP ) of 24.0 ± 0.37°C, at which point the electrical activity abruptly ceased. The maximum f PM at T BP was much higher [193 ± 21.0 beats per minute (bpm)] than the peak f SA (94.3 ± 6.0 bpm at 24.1°C) or peak f H (76.7 ± 2.4 at 15.7 ± 0.82°C) (P < 0.05). These findings strongly suggest that the frequency generator of the sinoatrial pacemaker cells does not limit f H at high temperatures in the brown trout in vivo., (Copyright © 2017 the American Physiological Society.)

Insights into the expression of pacemaker-specific markers in human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte subtypes can facilitate the enrichment and track differentiation and maturation of hiPSC-derived pacemaker-like cardiomyocytes. To date, no study has directly assessed gene expression in each pacemaker-, atria-, and ventricular-like cardiomyocyte subtype derived from hiPSCs since currently the subtypes of these immature cardiomyocytes can only be identified by action potential profiles. Traditional acquisition of action potentials using patch-clamp recordings renders the cells unviable for subsequent analysis. We circumvented these issues by acquiring the action potential profile of a single cell optically followed by assessment of protein expression through immunostaining in that same cell. Our same-single-cell analysis for the first time revealed expression of proposed pacemaker-specific markers-hyperpolarization-activated cyclic nucleotide-modulated (HCN)4 channel and Islet (Isl)1-at the protein level in all three hiPSC-derived cardiomyocyte subtypes. HCN4 expression was found to be higher in pacemaker-like hiPSC-derived cardiomyocytes than atrial- and ventricular-like subtypes but its downregulation over time in all subtypes diminished the differences. Isl1 expression in pacemaker-like hiPSC-derived cardiomyocytes was initially not statistically different than the contractile subtypes but did become statistically higher than ventricular-like cells with time. Our observations suggest that although HCN4 and Isl1 are differentially expressed in hiPSC-derived pacemaker-like relative to ventricular-like cardiomyocytes, these markers alone are insufficient in identifying hiPSC-derived pacemaker-like cardiomyocytes. Stem Cells 2016;34:2670-2680., (© 2016 AlphaMed Press.)

The specialised conducting tissues present in the ventricles are responsible for the fast distribution of the electrical impulse from the atrio-ventricular node to regions in the subendocardial myocardium. Characterisation of anatomical features of the specialised conducting tissues in the ventricles is highly challenging, in particular its most distal section, which is connected to the working myocardium via Purkinje-myocardial junctions. The goal of this work is to characterise the architecture of the distal section of the Purkinje network by differentiating Purkinje cells from surrounding tissue, performing a segmentation of Purkinje fibres at cellular scale, and mathematically describing its morphology and interconnections. Purkinje cells from rabbit hearts were visualised by confocal microscopy using wheat germ agglutinin labelling. A total of 16 3D stacks including labeled Purkinje cells were collected, and semi-automatically segmented. State-of-the-art graph metrics were applied to estimate regional and global features of the Purkinje network complexity. Two types of cell types, tubular and star-like, were characterised from 3D segmentations. The analysis of 3D imaging data confirms the previously suggested presence of two types of Purkinje-myocardium connections, a 2D interconnection sheet and a funnel one, in which the narrow side of a Purkinje fibre connect progressively to muscle fibres. The complex network analysis of interconnected Purkinje cells showed no small-world connectivity or assortativity properties. These results might help building more realistic computational PK systems at high resolution levels including different cell configurations and shapes. Better knowledge on the organisation of the network might help in understanding the effects that several treatments such as radio-frequency ablation might have when the PK system is disrupted locally., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The heart is an electrically conducting organ with networked bioelectric currents that transverse a large segment of interstitial space interspersed with the muscular parenchyma. Non-excitable connective cells in the interstitial space contributed importantly to many structural, biochemical, and physiological activities of cardiac homeostasis. However, contribution of interstitial cells in the cardiac niche has long been neglected. Telocyte is recently recognized as a distinct class of interstitial cell that resides in a wide array of tissues including in the epicardium, myocardium, and endocardium of the heart. They are increasingly described to conduct ionic currents that may have significant implications in bioelectric signaling. In this review, we highlight the significance of telocytes in such connectivity and conductivity within the interstitial bioelectric network in tissue homeostasis.

Models of electrical activity in cardiac cells have become important research tools as they can provide a quantitative description of detailed and integrative physiology. However, cardiac cell models have many parameters, and how uncertainties in these parameters affect the model output is difficult to assess without undertaking large numbers of model runs. In this study we show that a surrogate statistical model of a cardiac cell model (the Luo-Rudy 1991 model) can be built using Gaussian process (GP) emulators. Using this approach we examined how eight outputs describing the action potential shape and action potential duration restitution depend on six inputs, which we selected to be the maximum conductances in the Luo-Rudy 1991 model. We found that the GP emulators could be fitted to a small number of model runs, and behaved as would be expected based on the underlying physiology that the model represents. We have shown that an emulator approach is a powerful tool for uncertainty and sensitivity analysis in cardiac cell models.

Specialized myocytes of the cardiac conduction system (CCS) are essential to coordinate sequential contraction of cardiac atria and ventricles. Anomalies of the CCS can result in lethal cardiac arrhythmias, including sick sinus syndrome and atrial or ventricular fibrillation. To develop future therapies and regenerative medicine aimed at cardiac arrhythmias, it is important to understand formation and function of distinct components of the CCS. Essential to this understanding is the development of CCS-specific markers. In this review, we briefly summarize available mouse models of CCS markers and focus on those involving the hyperpolarization cation-selective nucleotide-gated cation channel, HCN4, which selectively marks all components of the specialized CCS in adult heart. Recent studies have revealed, however, that HCN4 expression during development is highly dynamic in cardiac precursors. These studies have offered insights into the contributions of the first and second heart field to myocyte and conduction system lineages and suggested the timing of allocation of specific conduction system precursors during development. Altogether, they have highlighted the utility of HCN4 as a cell surface marker for distinct components of the CCS at distinct stages of development, which can be utilized to facilitate purification and characterization of CCS precursors in mouse and human model systems and pave the way for regenerative therapies., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Impulse propagation in biological tissues is known to be modulated by structural heterogeneity. In cardiac muscle, improved understanding on how this heterogeneity influences electrical spread is key to advancing our interpretation of dispersion of repolarization. We propose fractional diffusion models as a novel mathematical description of structurally heterogeneous excitable media, as a means of representing the modulation of the total electric field by the secondary electrical sources associated with tissue inhomogeneities. Our results, analysed against in vivo human recordings and experimental data of different animal species, indicate that structural heterogeneity underlies relevant characteristics of cardiac electrical propagation at tissue level. These include conduction effects on action potential (AP) morphology, the shortening of AP duration along the activation pathway and the progressive modulation by premature beats of spatial patterns of dispersion of repolarization. The proposed approach may also have important implications in other research fields involving excitable complex media.

Anchoring cell junctions are integral in maintaining electro-mechanical coupling of ventricular working cardiomyocytes; however, their role in cardiomyocytes of the cardiac conduction system (CCS) remains less clear. Recent studies in genetic mouse models and humans highlight the appearance of these cell junctions alongside gap junctions in the CCS and also show that defects in these structures and their components are associated with conduction impairments in the CCS. Here we outline current evidence supporting an integral relationship between anchoring and gap junctions in the CCS. Specifically we focus on (1) molecular and ultrastructural evidence for cell-cell junctions in specialized cardiomyocytes of the CCS, (2) genetic mouse models specifically targeting cell-cell junction components in the heart which exhibit CCS conduction defects and (3) human clinical studies from patients with cell-cell junction-based diseases that exhibit CCS electrophysiological defects.

Rationale: Tight control of cardiomyocyte proliferation is essential for the formation of four-chambered heart. Although human mutation of NKX2-5 is linked to septal defects and atrioventricular conduction abnormalities, early lethality and hemodynamic alteration in the mutant models have caused controversy as to whether Nkx2-5 regulates cardiomyocyte proliferation., Objective: In this study, we circumvented these limitations by atrial-restricted deletion of Nkx2-5., Method and Results: Atrial-specific Nkx2-5 mutants died shortly after birth with hyperplastic working myocytes and conduction system including two nodes and internodal tracts. Multicolor reporter analysis revealed that Nkx2-5-null cardiomyocytes displayed clonal proliferative activity throughout the atria, indicating the suppressive role of Nkx2-5 in cardiomyocyte proliferation after chamber ballooning stages. Transcriptome analysis revealed that aberrant activation of Notch signaling underlies hyperproliferation of mutant cardiomyocytes, and forced activation of Notch signaling recapitulates hyperproliferation of working myocytes but not the conduction system., Conclusions: Collectively, these data suggest that Nkx2-5 regulates the proliferation of atrial working and conduction myocardium in coordination with Notch pathway.

Neurons generated from pluripotent stem cells (PSCs) self-organize into functional neuronal assemblies in vitro, generating synchronous network activities. Intriguingly, PSC-derived neuronal assemblies develop spontaneous activities that are independent of external stimulation, suggesting the presence of thus far undetected intrinsically active neurons (IANs). Here, by using mouse embryonic stem cells, we provide evidence for the existence of IANs in PSC-neuronal networks based on extracellular multielectrode array and intracellular patch-clamp recordings. IANs remain active after pharmacological inhibition of fast synaptic communication and possess intrinsic mechanisms required for autonomous neuronal activity. PSC-derived IANs are functionally integrated in PSC-neuronal populations, contribute to synchronous network bursting, and exhibit pacemaker properties. The intrinsic activity and pacemaker properties of the neuronal subpopulation identified herein may be particularly relevant for interventions involving transplantation of neural tissues. IANs may be a key element in the regulation of the functional activity of grafted as well as preexisting host neuronal networks.

Numerical integration of mathematical models of heart cell electrophysiology provides an important computational tool for studying cardiac arrhythmias, but the abundance of available models complicates selecting an appropriate model. We study the behavior of two recently published models of human ventricular action potentials, the Grandi-Pasqualini-Bers (GPB) and the O'Hara-Virág-Varró-Rudy (OVVR) models, and compare the results with four previously published models and with available experimental and clinical data. We find the shapes and durations of action potentials and calcium transients differ between the GPB and OVVR models, as do the magnitudes and rate-dependent properties of transmembrane currents and the calcium transient. Differences also occur in the steady-state and S1-S2 action potential duration and conduction velocity restitution curves, including a maximum conduction velocity for the OVVR model roughly half that of the GPB model and well below clinical values. Between single cells and tissue, both models exhibit differences in properties, including maximum upstroke velocity, action potential amplitude, and minimum diastolic interval. Compared to experimental data, action potential durations for the GPB and OVVR models agree fairly well (although OVVR epicardial action potentials are shorter), but maximum slopes of steady-state restitution curves are smaller. Although studies show alternans in normal hearts, it occurs only in the OVVR model, and only for a narrow range of cycle lengths. We find initiated spiral waves do not progress to sustained breakup for either model. The dominant spiral wave period of the GPB model falls within clinically relevant values for ventricular tachycardia (VT), but for the OVVR model, the dominant period is longer than periods associated with VT. Our results should facilitate choosing a model to match properties of interest in human cardiac tissue and to replicate arrhythmia behavior more closely. Furthermore, by indicating areas where existing models disagree, our findings suggest avenues for further experimental work.

Rationale: To date, there has been no specific marker of the first heart field to facilitate understanding of contributions of the first heart field to cardiac lineages. Cardiac arrhythmia is a leading cause of death, often resulting from abnormalities in the cardiac conduction system (CCS). Understanding origins and identifying markers of CCS lineages are essential steps toward modeling diseases of the CCS and for development of biological pacemakers., Objective: To investigate HCN4 as a marker for the first heart field and for precursors of distinct components of the CCS, and to gain insight into contributions of first and second heart lineages to the CCS., Methods and Results: HCN4CreERT2, -nuclear LacZ, and -H2BGFP mouse lines were generated. HCN4 expression was examined by means of immunostaining with HCN4 antibody and reporter gene expression. Lineage studies were performed using HCN4CreERT2, Isl1Cre, Nkx2.5Cre, and Tbx18Cre, coupled to coimmunostaining with CCS markers. Results demonstrated that, at cardiac crescent stages, HCN4 marks the first heart field, with HCN4CreERT2 allowing assessment of cell fates adopted by first heart field myocytes. Throughout embryonic development, HCN4 expression marked distinct CCS precursors at distinct stages, marking the entire CCS by late fetal stages. We also noted expression of HCN4 in distinct subsets of endothelium at specific developmental stages., Conclusions: This study provides insight into contributions of first and second heart lineages to the CCS and highlights the potential use of HCN4 in conjunction with other markers for optimization of protocols for generation and isolation of specific conduction system precursors.

Aims: Despite recognition that understanding gross anatomy of the right atrium is important in the era of invasive electrophysiology, areas such as the right atrial appendage (RAA) wall or the vestibule are not fully appreciated. The aim of this study was to conduct an anatomical assessment focusing on these structures to gain further insights into electrophysiological procedures., Methods: Forty-four normal human hearts were examined macro- and microscopically., Results: Inside the RAA, two prominent muscle bundles; the crista terminalis (CT) and the sagittal bundle (SB) were identified. The medial wall at which the CT originated from and its surrounding area was the thickest part adjacent anteriorly to the thin aortic mound, suggesting non-uniform wall thickness of this area. Histological sections revealed that myocardial strands of the SB connected the CT and RAA tip, implying preferential anterior conduction of the sinus impulse. The vestibule had a thin myocardium with extensive fat covering along the epicardial side of the tricuspid annulus. The proximal portion of the right coronary artery (RCA) was relatively distant from the annulus, followed by gradual shortening of the distance from the endocardium to the RCA, which led to a very close relationship (<3.0 mm) at the inferior annulus., Conclusion: Non-uniform wall thickness and muscle fibre orientation in the RAA should be taken into consideration during lead/catheter positioning. The RCA proximity in the inferior portion of the vestibule and the deeper fatty plane of the anterior atrioventricular groove are important anatomical features relevant to accessory pathway ablation.

Thermal effects affecting spatiotemporal behavior of cardiac tissue are discussed by relating temperature variations to proarrhythmic dynamics in the heart. By introducing a thermoelectric coupling in a minimal model of cardiac tissue, we are able to reproduce experimentally measured dynamics obtained simultaneously from epicardial and endocardial canine right ventricles at different temperatures. A quantitative description of emergent proarrhythmic properties of restitution, conduction velocity, and alternans regimes as a function of temperature is presented. Complex discordant alternans patterns that enhance tissue dispersion consisting of one wave front and three wave backs are described in both simulations and experiments. Possible implications for model generalization are finally discussed.

Various forms of heart disease are associated with remodeling of the heart muscle, which results in a perturbation of cell-to-cell electrical coupling. These perturbations may alter the trajectory of spiral wave drift in the heart muscle. We investigate the effect of spatially extended inhomogeneity of transverse cell coupling on the spiral wave trajectory using a simple active media model. The spiral wave was either attracted or repelled from the center of inhomogeneity as a function of cell excitability and gradient of the cell coupling. High levels of excitability resulted in an attraction of the wave to the center of inhomogeneity, whereas low levels resulted in an escape and termination of the spiral wave. The spiral wave drift velocity was related to the gradient of the coupling and the initial position of the wave. In a diseased heart, a region of altered transverse coupling corresponds with local gap junction remodeling that may be responsible for stabilization-destabilization of spiral waves and hence reflect potentially important targets in the treatment of heart arrhythmias.

Recent progress in diffusion imaging has lead to in-vivo acquisitions of fiber orientation data in the beating heart. Current methods are however limited in resolution to a few short-axis slices. For this particular application and others where the diffusion volume is subsampled, partial or even damaged, the reconstruction of a complete volume can be challenging. To address this problem, we present two complementary methods for fiber reconstruction from sparse orientation measurements, both of which derive from second-order properties related to fiber curvature as described by Maurer-Cartan connection forms. The first is an extrinsic partial volume reconstruction method based on principal component analysis of the connection forms and is best put to use when dealing with highly damaged or sparse data. The second is an intrinsic method based on curvilinear interpolation of the connection forms on ellipsoidal shells and is advantageous when more slice data becomes available. Using a database of 8 cardiac rat diffusion tensor images we demonstrate that both methods are able to reconstruct complete volumes to good accuracy and lead to low reconstruction errors.

Myocardial development is regulated by an elegantly choreographed ensemble of signaling events mediated by a multitude of intermediates that take a variety of forms. Cellular differentiation and maturation are a subset of vertically integrated processes that extend over several spatial and temporal scales to create a well-defined collective of cells that are able to function cooperatively and reliably at the organ level. Early efforts to understand the molecular mechanisms of cardiomyocyte fate determination focused primarily on genetic and chemical mediators of this process. However, increasing evidence suggests that mechanical interactions between the extracellular matrix (ECM) and cell surface receptors as well as physical interactions between neighboring cells play important roles in regulating the signaling pathways controlling the developmental processes of the heart. Interdisciplinary efforts have made it apparent that the influence of the ECM on cellular behavior occurs through a multitude of physical mechanisms, such as ECM boundary conditions, elasticity, and the propagation of mechanical signals to intracellular compartments, such as the nucleus. In addition to experimental studies, a number of mathematical models have been developed that attempt to capture the interplay between cells and their local microenvironment and the influence these interactions have on cellular self-assembly and functional behavior. Nevertheless, many questions remain unanswered concerning the mechanism through which physical interactions between cardiomyocytes and their environment are translated into biochemical cellular responses and how these signaling modalities can be utilized in vitro to fabricate myocardial tissue constructs from stem cell-derived cardiomyocytes that more faithfully represent their in vivo counterpart. These studies represent a broad effort to characterize biological form as a conduit for information transfer that spans the nanometer length scale of proteins to the meter length scale of the patient and may yield new insights into the contribution of mechanotransduction into heart development and disease.

Calcium influx is required for the pituitary adenylyl cyclase activating polypeptide (PACAP)-induced increase in guinea pig cardiac neuron excitability, noted as a change from a phasic to multiple action potential firing pattern. Intracellular recordings indicated that pretreatment with the nonselective cationic channel inhibitors, 2-aminoethoxydiphenylborate (2-APB), 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole HCl (SKF 96365), and flufenamic acid (FFA) reduced the 20-nM PACAP-induced excitability increase. Additional experiments tested whether 2-APB, FFA, and SKF 96365 could suppress the increase in excitability by PACAP once it had developed. The increased action potential firing remained following application of 2-APB but was diminished by FFA. SKF 96365 transiently depressed the PACAP-induced excitability increase. A decrease and recovery of action potential amplitude paralleled the excitability shift. Since semiquantitative PCR indicated that cardiac neurons express TRPC subunit transcripts, we hypothesize that PACAP activates calcium-permeable, nonselective cationic channels, which possibly are members of the TRPC family. Our results are consistent with calcium influx being required for the initiation of the PACAP-induced increase in excitability, but suggest that it may not be required to sustain the peptide effect. The present results also demonstrate that nonselective cationic channel inhibitors could have other actions, which might contribute to the inhibition of the PACAP-induced excitability increase.

In this review, we discuss recent studies on the mechanosensitive morphology and function of cardiomyocytes derived from embryos and neonates. For early cardiomyocytes cultured on substrates of various stiffnesses, contractile function as measured by force production, work output and calcium handling is optimized when the culture substrate stiffness mimics that of the tissue from which the cells were obtained. This optimal contractile function corresponds to changes in sarcomeric protein conformation and organization that promote contractile ability. In light of current models for myofibillogenesis, a recent mathematical model of striation and alignment on elastic substrates helps to illuminate how substrate stiffness modulates early myofibril formation and organization. During embryonic heart formation and maturation, cardiac tissue mechanics change dynamically. Experiments and models highlighted here have important implications for understanding cardiomyocyte differentiation and function in development and perhaps in regeneration processes.

In cat atrial myocytes, β-adrenergic receptor (β-AR) stimulation exerts profound effects on excitation-contraction coupling and cellular Ca(2+) cycling that are mediated by β(1)- and β(2)-AR subtypes coupled to G proteins (G(s) and G(i)). In this study, we determined the effects of β-AR stimulation on pacing-induced Ca(2+) alternans. Ca(2+) alternans was recorded from single cat atrial myocytes with the fluorescent Ca(2+) indicator indo-1. Stable Ca(2+) alternans occurred at an average pacing frequency of 1.7 Hz at room temperature with a mean alternans ratio of 0.43. Nonselective β-AR stimulation as well as selective stimulation of β(1)/G(s), β(2)/G(s) + G(i), and β(2)/G(s) coupled pathways all abolished pacing-induced Ca(2+) alternans. β(1)-AR stimulation abolished alternans through stimulation of PKA and Ca(2+)/calmodulin-dependent protein kinase II, whereas β(2)-AR stimulation exclusively involved PKA and was mediated via G(s), whereas a known second pathway in cat atrial myocytes acting through G(i) and nitric oxide production was not involved in alternans regulation. Inhibition of various mitochondrial functions (dissipation of the mitochondrial membrane potential or inhibition of mitochondrial F(1)/F(0)-ATP synthase, mitochondrial Ca(2+) uptake via the mitochondrial Ca(2+) uniporter, and Ca(2+) extrusion via mitochondrial Na(+)/Ca(2+) exchange) enhanced Ca(2+) alternans; however, β-AR stimulation still abrogated alternans, provided that sufficient cellular ATP was available. Selective inhibition of mitochondrial or glycolytic ATP production did not prevent β-AR stimulation from abolishing Ca(2+) alternans. However, when both ATP sources were depleted, β-AR stimulation failed to decrease Ca(2+) alternans. These results indicate that in atrial myocytes, β-AR stimulation protects against pacing-induced alternans by acting through parallel and complementary signaling pathways.

Local anesthetic toxicity includes a decrease in ventricular conduction velocity and a decrease in myocardial contractile force. We tested the hypothesis that, like conduction, contraction was stereoselectively impaired by bupivacaine. We compared R(+) and S(-) bupivacaine to S(+) and R(-) RAC 109 in order to study the effects of hydrophobicity and ionization. We measured the changes in QRS duration and developed pressure in isolated perfused rabbit hearts. Binding of bupivacaine and RAC 109 to the ryanodine receptor was measured. The effect on cell shortening and relenghtening was measured on isolated rabbit cardiomyocytes. A mixed-effect pharmacodynamic model was used. The decrease in conduction velocity induced by the molecules was markedly stereospecific. All local anesthetics decreased ventricular velocity in a stereospecific manner with a RAC I(+)/II(-) and bupivacaine R(+)/S(-) potency ratio of maximum effect of 1.7 and 2.25 respectively. Contractility decreased in a dose dependent manner but this negative inotropic effect was not stereospecific with a C50 (concentration leading to half maximum effect) of 30 and 23 μM for RAC and bupivacaine respectively. The two drugs exhibited two-site binding to ryanodyne that may partly explain the observed effect. An effect on relaxation was observed only at very high concentrations. In conclusion, bupivacaine, a long acting local anesthetic, decreases ventricular conduction in a stereospecific manner, and decreases contractility non-stereospecifically., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The atrial pacemaker complex is responsible for the initiation and early propagation of cardiac impulses. Optical coherence tomography (OCT), a nondestructive imaging modality with spatial resolutions of ∼1 to 15 μm, can be used to identify unique fiber orientation patterns in this region of the heart. Functionally characterized canine sinoatrial nodes (SAN) (n=7) were imaged using OCT up to ∼1  mm below the endocardial tissue surface. OCT images were directly compared to their corresponding histological sections. Fiber orientation patterns unique to the crista terminalis (CT), SAN, and surrounding atrial myocardium were identified with dominant average fiber angles of 89 ± 12 deg, 110 ± 16 deg, and 95 ± 35 deg, respectively. Both the CT and surrounding atrial myocardium displayed predominantly unidirectionally based fiber orientation patterns within each specimen, whereas the SAN displayed an increased amount of fiber disarray manifested quantitatively as a significantly greater standard deviation in fiber angle distribution within specimens [33 ± 7 deg versus 23 ± 5 deg, atrium (p=0.02); 18 ± 3 deg, CT (p=0.0003)]. We also identified unique, local patterns of fiber orientation specific to the functionally characterized block zone. We demonstrate the ability of OCT in detecting components of the atrial pacemaker complex which are intimately involved in both normal and abnormal cardiac conduction.

Histochemistry for acetylcholinesterase was used to determine the distribution of intracardiac neurons in the frog Rana temporaria. Seventy-nine intracardiac neurons from 13 frogs were labelled iontophoretically by the intracellular markers Alexa Fluor 568 and Lucifer Yellow CH to determine their structure and projections. Total neuronal number per frog heart was (Mean ± SE) 1374 ± 56. Largest collections of neurons were found in the interatrial septum (46%), atrioventricular junction (25%) and venal sinus (12%). Among the intracellularly labelled neurons, we found the cells of unipolar (71%), multipolar (20%) and bipolar (9%) types. Multiple processes originated from the neuron soma, hillock and proximal axon. These processes projected onto adjacent neuron somata and cardiac muscle fibers within the interatrial septum. Average total length of the processes from proximal axon was 348 ± 50 μm. Average total length of processes from soma and hillock was less, 118 ± 27 μm and 109 ± 24 μm, respectively. The somata of 59% of neurons had bubble- or flake-shaped extensions. Most neurons from the major nerves in the interatrial septum sent their axons towards the ventricle. In contrast, most neurons from the ventral part of the interatrial septum sent their axons towards the atria. Our findings contradict to a view that the frog intracardiac ganglia contain only non-dendritic neurons of the unipolar type. We conclude that the frog intracardiac neurons are structurally complex and diverse. This diversity may account for the complicated integrative functions of the frog intrinsic cardiac ganglia., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Methods for the electrical inhibition of cardiac excitation have long been sought to control excitability and conduction, but to date remain largely impractical. High-amplitude alternating current (AC) stimulation has been known to extend cardiac action potentials (APs), and has been recently exploited to terminate reentrant arrhythmias by producing reversible conduction blocks. Yet, low-amplitude currents at similar frequencies have been shown to entrain cardiac tissues by generation of repetitive APs, leading in some cases to ventricular fibrillation and hemodynamic collapse in vivo. Therefore, an inhibition method that does not lead to entrainment - irrespective of the stimulation amplitude (bound to fluctuate in an in vivo setting) - is highly desirable., Methodology/principal Findings: We investigated the effects of broader amplitude and frequency ranges on the inhibitory effects of extracellular AC stimulation on HL-1 cardiomyocytes cultured on microelectrode arrays, using both sinusoidal and square waveforms. Our results indicate that, at sufficiently high frequencies, cardiac tissue exhibits a binary response to stimulus amplitude with either prolonged APs or no effect, thereby effectively avoiding the risks of entrainment by repetitive firing observed at lower frequencies. We further demonstrate the ability to precisely define reversible local conduction blocks in beating cultures without influencing the propagation activity in non-blocked areas. The conduction blocks were spatiotemporally controlled by electrode geometry and stimuli duration, respectively, and sustainable for long durations (300 s)., Conclusion/significance: Inhibition of cardiac excitation induced by high-frequency AC stimulation exhibits a binary response to amplitude above a threshold frequency, enabling the generation of reversible conduction blocks without the risks of entrainment. This inhibition method could yield novel approaches for arrhythmia modeling in vitro, as well as safer and more efficacious tools for in vivo cardiac mapping and radio-frequency ablation guidance applications.

The atrioventricular conduction axis, located in the septal component of the atrioventricular junctions, is arguably the most complex structure in the heart. It fulfils a multitude of functions, including the introduction of a delay between atrial and ventricular systole and backup pacemaking. Like any other multifunctional tissue, complexity is a key feature of this specialised tissue in the heart, and this complexity is both anatomical and electrophysiological, with the two being inextricably linked. We used quantitative PCR, histology and immunohistochemistry to analyse the axis from six human subjects. mRNAs for ~50 ion and gap junction channels, Ca(2+)-handling proteins and markers were measured in the atrial muscle (AM), a transitional area (TA), inferior nodal extension (INE), compact node (CN), penetrating bundle (PB) and ventricular muscle (VM). When compared to the AM, we found a lower expression of Na(v)1.5, K(ir)2.1, Cx43 and ANP mRNAs in the CN for example, but a higher expression of HCN1, HCN4, Ca(v)1.3, Ca(v)3.1, K(ir)3.4, Cx40 and Tbx3 mRNAs. Expression of some related proteins was in agreement with the expression of the corresponding mRNAs. There is a complex and heterogeneous pattern of expression of ion and gap junction channels and Ca(2+)-handling proteins in the human atrioventricular conduction axis that explains the function of this crucial pathway., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The gap junction and voltage-gated Na(+) channel play an important role in the action potential propagation. The purpose of this study was to elucidate the roles of subcellular Na(+) channel distribution in action potential propagation. To achieve this, we constructed the myocardial strand model, which can calculate the current via intercellular cleft (electric-field mechanism) together with gap-junctional current (gap-junctional mechanism). We conducted simulations of action potential propagation in a myofiber model where cardiomyocytes were electrically coupled with gap junctions alone or with both the gap junctions and the electric field mechanism. Then we found that the action potential propagation was greatly affected by the subcellular distribution of Na(+) channels in the presence of the electric field mechanism. The presence of Na(+) channels in the lateral membrane was important to ensure the stability of propagation under conditions of reduced gap-junctional coupling. In the poorly coupled tissue with sufficient Na(+) channels in the lateral membrane, the slowing of action potential propagation resulted from the periodic and intermittent dysfunction of the electric field mechanism. The changes in the subcellular Na(+) channel distribution might be in part responsible for the homeostatic excitation propagation in the diseased heart., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The presence of connective tissue as well as interstitial clefts forms a natural barrier to the electrical propagation in the heart. At a microscopic scale, such uncoupling structures change the pattern of the electrical conduction from uniform towards complex and may play a role in the genesis of cardiac arrhythmias. The anatomical diversity of conduction structures and their topology at a microscopic size scale is overwhelming for experimental techniques. Mathematical models have been often employed to study the behavior of the electrical propagation at a sub-cellular level. However, very fine and computationally expensive meshes are required to capture all microscopic details found in the cardiac tissue. In this work, we present a numerical technique based on the finite element method which allows to reproduce the effects of microscopic conduction barriers caused by the presence of uncoupling structures without actually resolving these structures in a high resolution mesh, thereby reducing the computational costs significantly.

Introduction: Computer simulations suggest that intercellular coupling is more robust than membrane excitability with regard to changes in and safety of conduction. Clinical studies indicate that SCN5A (excitability) and/or Connexin43 (Cx43, intercellular coupling) expression in heart disease is reduced by approximately 50%. In this retrospective study we assessed the effect of reduced membrane excitability or intercellular coupling on conduction in mouse models of reduced excitability or intercellular coupling., Methods and Results: Epicardial activation mapping of LV and RV was performed on Langendorff-perfused mouse hearts having the following: 1) Reduced excitability: Scn5a haploinsufficient mice; and 2) reduced intercellular coupling: Cx43(CreER(T)/fl) mice, uninduced (50% Cx43) or induced (10% Cx43) with Tamoxifen. Wild type (WT) littermates were used as control. Conduction velocity (CV) restitution and activation delay were determined longitudinal and transversal to fiber direction during S(1)S(1) pacing and S(1)S(2) premature stimulation until the effective refractory period. In both animal models, CV restitution and activation delay in LV were not changed compared to WT. In contrast, CV restitution decreased and activation delay increased in RV during conduction longitudinal but not transverse to fiber direction in Scn5a heterozygous animals compared to WT. In contrast, a 50% reduction of intercellular coupling did not affect either CV restitution or activation delay. A decrease of 90% Cx43, however, resulted in decreased CV restitution and increased activation delay in RV, but not LV., Conclusion: Reducing excitability but not intercellular coupling by 50% affects CV restitution and activation delay in RV, indicating a higher safety factor for intercellular coupling than excitability in RV.

The cardiac pacemaker controls the rhythmicity of heart contractions and can be substituted by a battery-operated device as a last resort. We created a genetically encoded, optically controlled pacemaker by expressing halorhodopsin and channelrhodopsin in zebrafish cardiomyocytes. Using patterned illumination in a selective plane illumination microscope, we located the pacemaker and simulated tachycardia, bradycardia, atrioventricular blocks, and cardiac arrest. The pacemaker converges to the sinoatrial region during development and comprises fewer than a dozen cells by the time the heart loops. Perturbation of the activity of these cells was entirely reversible, demonstrating the resilience of the endogenous pacemaker. Our studies combine optogenetics and light-sheet microscopy to reveal the emergence of organ function during development.

Background: Ion channels are key determinants for the function of excitable cells, but little is known about their role and involvement during cardiac development. Earlier work identified Ca(2+)-activated potassium channels of small and intermediate conductance (SKCas) as important regulators of neural stem cell fate. Here we have investigated their impact on the differentiation of pluripotent cells toward the cardiac lineage., Methods and Results: We have applied the SKCa activator 1-ethyl-2-benzimidazolinone on embryonic stem cells and identified this particular ion channel family as a new critical target involved in the generation of cardiac pacemaker-like cells: SKCa activation led to rapid remodeling of the actin cytoskeleton, inhibition of proliferation, induction of differentiation, and diminished teratoma formation. Time-restricted SKCa activation induced cardiac mesoderm and commitment to the cardiac lineage as shown by gene regulation, protein, and functional electrophysiological studies. In addition, the differentiation into cardiomyocytes was modulated in a qualitative fashion, resulting in a strong enrichment of pacemaker-like cells. This was accompanied by induction of the sino-atrial gene program and in parallel by a loss of the chamber-specific myocardium. In addition, SKCa activity induced activation of the Ras-Mek-Erk signaling cascade, a signaling pathway involved in the 1-ethyl-2-benzimidazolinone-induced effects., Conclusions: SKCa activation drives the fate of pluripotent cells toward mesoderm commitment and cardiomyocyte specification, preferentially into nodal-like cardiomyocytes. This provides a novel strategy for the enrichment of cardiomyocytes and in particular, the generation of a specific subtype of cardiomyocytes, pacemaker-like cells, without genetic modification.

Postnatal development of mammalian cardiomyocytes in the working myocardium is characterized by a near-complete translocation of both kinds of adhering junctions (AJs), i.e. desmosomes and fasciae adhaerentes (FAs), to the polar intercalated disk (ID) regions where they cluster, fuse and molecularly amalgamate to extended hybrid intercellular junction structures, the area composita (composite junction; AC). Using immunofluorescence and immunoelectron microscopy we now report that the AJ structures of the conduction system, in particular those of the Purkinje fiber cells of cows and sheep are fundamentally different. Here the numerous AJs remain in lateral connections with other conductive cells. Desmosomal or desmosome-like junctions can still be distinguished from FA junctions, and a third type of AJs can be identified which shows colocalization of desmosomal and FA proteins, i.e. an AC character. These results, together with demonstrations of other cell type cytoskeletal markers such as alpha-cardiac actin and desmin, support the concept that conductive cells are derived from embryonal cardiomyocytes and are arrested at an early stage of differentiation. We also show that the conductive cells have extended plasma membrane regions characterized by an exceptionally high proportion of junctions with desmosomal character and proteins, amounting to 50% and more, resulting in the highest desmosome protein packing so far described in non-epithelial cells. The relevance of these junctions for the formation, maintenance and functions of the conductive system is discussed, together with the conclusion that the desmosome-rich regions of conductive cells are among the most vulnerable sites for functional disorders caused by desmosomal protein mutations., (2010 Elsevier GmbH. All rights reserved.)

Background: Development of biological pacemaker is a potential treatment for bradyarrhythmias. Pacemaker cells could be extracted from differentiated embryonic stem (ES) cells based on their specific cell marker hyperpolarization-activated cyclic nucleotide-gated (HCN)4. The goal of this study was to develop a method of identification, isolation, and characterization of pacemaking cells derived from differentiated ES cells with GFP driven by HCN4 promoter., Methods and Results: Polymerase chain reaction (PCR) screening and southern blot analysis revealed that HCN4p-EGFP trans-gene was stably integrated into the chromosome of mouse AB1 ES cells. RT-PCR and immunostaining results showed similar expression of the specific cardiac pacemaker markers of the HCN4p-EGFP ES cells and its parental AB1 ES cell lines. Although HCN4p-EGFP trans-gene may have slight effect on the general mesodermal differentiation, it had no effect on the pluripotency of ES cells, on the transcription of cardiac specific factors and cardiac contractile proteins, and on the capability of ES cells to differentiate into pacemaker cells. Electrophysiological study indicated that HCN4p-GFP-positive cells revealed the spontaneous action potential, which was slowed by the treatment with 2 mM Cs(+), and expressed the hyperpolarization-activeted cation current I(f) encoded by HCN4 gene., Conclusion: By the approach of using stable transfectant of HCN4p-EGFP gene, the identification, isolation, and characterization of ES cell-derived pacemaking cells could be carried out.

Ventricular activation of the mouse heart differs significantly compared to activation in larger mammals. Knowledge of structural and functional characteristics of laboratory animals is essential for evaluation of results obtained from experiments. The present study was performed to evaluate whether the different pattern of activation is common to small rodents or unique for mice. Hearts of adult Wistar rats were isolated and Langendorff perfused. After removing the right and left ventricular free wall, extracellular activity of the septum and bundle branches (BB) was determined using a multi-terminal electrode harboring 247 terminals. Immunolabeling on cryosections was performed to assess expression and distribution of the gap junction proteins Connexin40 (Cx40), Cx43, Cx45, contractile (Desmin, alpha-actinin) and intercalated disk-related (N-cadherin, beta-catenin) proteins. Collagen distribution was assessed by Sirius Red staining. Reconstruction of the left and right bundle branch (LBB and RBB) using immuno-labeling revealed that the LBB spreads all over the septal surface. The RBB too is broad, albeit to a lesser extend than LBB. A sheet of connective tissue electrically separates the common bundle and proximal BB from the septal working myocardium. Immunolabeling revealed clear differences between the conduction system and the working myocardium with respect to expression level and distribution of the different proteins analyzed. The morphological organization of the area resulted in an electrical activation pattern of the septum comparable to what is common in larger mammals: earliest activation at the midseptum via the bundle branches. From our data we conclude that the pattern of ventricular activation in the rat heart and the structure of the conduction system fit to data described for larger mammals and differ from the different pattern previously found in mouse heart.