Cardiac Electrophysiology Research Research Articles

The potential of artificial intelligence to revolutionize health care delivery, research, and education in cardiac electrophysiology.

The field of electrophysiology (EP) has benefited from numerous seminal innovations and discoveries that have enabled clinicians to deliver therapies and interventions that save lives and promote quality of life. The rapid pace of innovation in EP may be hindered by several challenges including the aging population with increasing morbidity, the availability of multiple costly therapies that, in many instances, confer minor incremental benefit, the limitations of healthcare reimbursement, the lack of response to therapies by some patients, and the complications of the invasive procedures performed. To overcome these challenges and continue on a steadfast path of transformative innovation, the EP community must comprehensively explore how artificial intelligence (AI) can be applied to healthcare delivery, research, and education and consider all opportunities in which AI can catalyze innovation; create workflow, research, and education efficiencies; and improve patient outcomes at a lower cost. In this white paper, we define AI and discuss the potential of AI to revolutionize the EP field. We also address the requirements for implementing, maintaining, and enhancing quality when using AI and consider ethical, operational, and regulatory aspects of AI implementation. This manuscript will be followed by several perspective papers that will expand on some of these topics.

Defining cardiac stability during Langendorff perfusion

The Langendorff heart preparation is a valuable ex vivo technique for studying cardiac electrophysiology. For the past century it has been used in toxicology studies, drug testing, and disease modeling and has contributed significantly to understanding cardiac physiology, development, and pathological states. This study aimed to investigate the stability and reproducibility of the modern Langendorff heart preparation under various experimental conditions and to establish cardiac metric standards with corresponding exclusion criteria. Isolated hearts were extracted from adult Sprague Dawley rats and retrograde perfused with a modified Krebs-Henseleit buffer. A cohort of isolated hearts were used to establish a control baseline of multiple physiological parameters measured over time (e.g., electrocardiogram, flow rate, electrophysiology, electrical and calcium dynamics). These control baseline values were then compared to hearts exposed to the most common media additives in Langendorff studies (e.g., blebbistatin(−/−), bovine serum albumin, and voltage and calcium indicator dye). Over a three-hour period, cardiac metrics were monitored in stable Langendorff-perfused hearts. The addition of blebbistatin, DMSO or dyes did not change EP or ECG measurements as compared to control baseline. The most significant drift in all the EP values was ventricular effective refractory period (VERP). With or without blebbistatin, VERP increased from 40 to 60ms over the course of three hours. In that time heart rate and flow also decreased slightly. Blebbistatin was associated with an increased action potential duration but did not alter calcium transient duration. It was also observed that 1% circulating bovine serum albumin quenched the rh237 voltage dye signal 80% as measured by signal to noise. Constant pressure Langendorff-perfusion with a crystalloid buffer provides a reliable and reproducible model for cardiac electrophysiology research with a few caveats. The total length of study should be carefully considered to account for adaptations in cardiac physiology during longer perfusion times. As an excitation-contraction uncoupler, blebbistatin reduces motion artifact but delays action potential duration. Although BSA is used to minimize cardiac edema, it interferes with membrane bound voltage dyes. This study sets physiological parameters as a useful guide for future cardiac studies that use the isolated heart as a research model. American Physiological Society, R01HD108839 to N.G.P. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Differential effects of mechano-electric feedback mechanisms on whole-heart activation, repolarization, and tension.

The human heart is subject to highly variable amounts of strain during day-to-day activities and needs to adapt to a wide range of physiological demands. This adaptation is driven by an autoregulatory loop that includes both electrical and the mechanical components. In particular, mechanical forces are known to feed back into the cardiac electrophysiology system, which can result in pro- and anti-arrhythmic effects. Despite the widespread use of computational modelling and simulation for cardiac electrophysiology research, the majority of in silico experiments ignore this mechano-electric feedback entirely due to the high computational cost associated with solving cardiac mechanics. In this study, we therefore use an electromechanically coupled whole-heart model to investigate the differential and combined effects of electromechanical feedback mechanisms with a focus on their physiological relevance during sinus rhythm. In particular, we consider troponin-bound calcium, the effect of deformation on the tissue diffusion tensor, and stretch-activated channels. We found that activation of the myocardium was only significantly affected when including deformation into the diffusion term of the monodomain equation. Repolarization, on the other hand, was influenced by both troponin-bound calcium and stretch-activated channels and resulted in steeper repolarization gradients in the atria. The latter also caused afterdepolarizations in the atria. Due to its central role for tension development, calcium bound to troponin affected stroke volume and pressure. In conclusion, we found that mechano-electric feedback changes activation and repolarization patterns throughout the heart during sinus rhythm and lead to a markedly more heterogeneous electrophysiological substrate. KEY POINTS: The electrophysiological and mechanical function of the heart are tightly interrelated by excitation-contraction coupling (ECC) in the forward direction and mechano-electric feedback (MEF) in the reverse direction. While ECC is considered in many state-of-the-art computational models of cardiac electromechanics, less is known about the effect of different MEF mechanisms. Accounting for calcium bound to troponin increases stroke volume and delays repolarization. Geometry-mediated MEF leads to more heterogeneous activation and repolarization with steeper gradients. Both effects combine in an additive way. Non-selective stretch-activated channels as an additional MEF mechanism lead to heterogeneous diastolic transmembrane voltage, higher developed tension and delayed repolarization or afterdepolarizations in highly stretched parts of the atria. The differential and combined effects of these three MEF mechanisms during sinus rhythm activation in a human four-chamber heart model may have implications for arrhythmogenesis, both in terms of substrate (repolarization gradients) and triggers (ectopy).

OpenEP-Py and OpenEP-GUI: An interactive platform for the visualisation and analysis of clinical and simulated cardiac electrophysiology data

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): 1) King’s Health Partnership Research & Development Challenge Fund Award (funded through Medical Research Council, UK) 2) British Heart Foundation, UK Background Electroanatomic mapping data is typically stored in proprietary formats and is difficult to access and analyse. This presents a barrier to electrophysiology research. OpenEP has been developed over the past decade to address this issue, with the first public release of the software in late 2020. OpenEP provides a standard format for storing electrophysiology data as well as an extensive suite of analysis tools. However, the current implementation of OpenEP has several limitations. Firstly, it is written in Matlab and thus not accessible to researchers without a Matlab license. Secondly, it lacks a graphical interface mandating a level of programming expertise to use. Finally, it lacks interfaces for computational modelling data, limiting its use to clinical rather than simulated data. Purpose In this work we address these limitations by developing (1) a Python-based implementation of OpenEP; (2) a graphical interface for interacting with OpenEP data and (3) interfaces for computational modelling environments. Methods Following review of the OpenEP source code, we developed use-case documentation for each OpenEP function and the proposed graphical user interface. By inspection of simulation outputs from an open-source simulation environment (openCARP) we designed new interfaces for modelling data. The implementation of OpenEP-py and OpenEP-GUI is based on the standard scientific Python stack with a PyQt front-end. Results OpenEP-Py is an open-source Python implementation for manipulation of electrophysiology data. The feature set is under active development and aims to match that of the Matlab implementation of OpenEP. In addition, OpenEP-Py can read data from cardiac electrophysiology simulations. The simulated data is stored in the standard OpenEP data structure, meaning analysis tools for clinical data can be used on the simulated data. Further, clinical data can be exported into the openCARP format, streamlining the creation of patient-specific models for simulations. Finally, because the software is open-source, OpenEP provides a transparent process for analysing data and thereby improves the reproducibility of studies. OpenEP-GUI (Figure 1) is a cross-platform desktop application for the visualisation and analysis of clinical and simulated cardiac electrophysiology data. It uses OpenEP-Py for loading, visualising, analysis and exporting electroanatomic mapping data. Multiple cases can be loaded via the System Manager, facilitating qualitative and quantitative comparison between cases. OpenEP-Py and OpenEP-GUI are licensed under the GNU-GPL v3 [WS1] and available online. Conclusions OpenEP-Py and OpenEP-GUI provide a simple yet effective way to perform cardiac electrophysiology research. The software is both open-source and under active development. We welcome feedback, feature requests, and contributions from the wider electrophysiology research community.

A Review of Healthy and Fibrotic Myocardium Microstructure Modeling and Corresponding Intracardiac Electrograms.

Computational simulations of cardiac electrophysiology provide detailed information on the depolarization phenomena at different spatial and temporal scales. With the development of new hardware and software, in silico experiments have gained more importance in cardiac electrophysiology research. For plane waves in healthy tissue, in vivo and in silico electrograms at the surface of the tissue demonstrate symmetric morphology and high peak-to-peak amplitude. Simulations provided insight into the factors that alter the morphology and amplitude of the electrograms. The situation is more complex in remodeled tissue with fibrotic infiltrations. Clinically, different changes including fractionation of the signal, extended duration and reduced amplitude have been described. In silico, numerous approaches have been proposed to represent the pathological changes on different spatial and functional scales. Different modeling approaches can reproduce distinct subsets of the clinically observed electrogram phenomena. This review provides an overview of how different modeling approaches to incorporate fibrotic and structural remodeling affect the electrogram and highlights open challenges to be addressed in future research.

Novel Optics-Based Approaches for Cardiac Electrophysiology: A Review.

Optical techniques for recording and manipulating cellular electrophysiology have advanced rapidly in just a few decades. These developments allow for the analysis of cardiac cellular dynamics at multiple scales while largely overcoming the drawbacks associated with the use of electrodes. The recent advent of optogenetics opens up new possibilities for regional and tissue-level electrophysiological control and hold promise for future novel clinical applications. This article, which emerged from the international NOTICE workshop in 20181, reviews the state-of-the-art optical techniques used for cardiac electrophysiological research and the underlying biophysics. The design and performance of optical reporters and optogenetic actuators are reviewed along with limitations of current probes. The physics of light interaction with cardiac tissue is detailed and associated challenges with the use of optical sensors and actuators are presented. Case studies include the use of fluorescence recovery after photobleaching and super-resolution microscopy to explore the micro-structure of cardiac cells and a review of two photon and light sheet technologies applied to cardiac tissue. The emergence of cardiac optogenetics is reviewed and the current work exploring the potential clinical use of optogenetics is also described. Approaches which combine optogenetic manipulation and optical voltage measurement are discussed, in terms of platforms that allow real-time manipulation of whole heart electrophysiology in open and closed-loop systems to study optimal ways to terminate spiral arrhythmias. The design and operation of optics-based approaches that allow high-throughput cardiac electrophysiological assays is presented. Finally, emerging techniques of photo-acoustic imaging and stress sensors are described along with strategies for future development and establishment of these techniques in mainstream electrophysiological research.

A Complete and Low-Cost Cardiac Optical Mapping System in Translational Animal Models.

Clinicians, biologists, physicists, engineers, and computer scientists are coming together to better understand heart disease, which is currently the leading cause of death globally. Optical mapping, a high-speed fluorescence imaging technique that visualizes and measures key cardiac parameters such as action potentials, cytosolic calcium transients, and fibrillation dynamics, is a core research tool that has arisen from such interdisciplinary collaborations. In an effort to broaden its use, especially among clinical scientists and students, we developed a complete and low-cost optical mapping system, including a constant-flow Langendorff perfusion system, which minimizes the economic threshold to widespread use of this powerful tool in cardiac electrophysiology research. The system described here provides high spatiotemporal resolution data about action potentials, intracellular calcium transients and fibrillation wave dynamics in isolated Langendorff-perfused hearts (pigs and rabbits), relevant for translational research. All system components and software elements are fully disclosed with the aim of increasing the use of this affordable and highly versatile tool among clinicians, basic scientists and students wishing to tackle their own research questions with their own customizable systems.

ESC working group on cardiac cellular electrophysiology position paper: relevance, opportunities, and limitations of experimental models for cardiac electrophysiology research.

Cardiac arrhythmias are a major cause of death and disability. A large number of experimental cell and animal models have been developed to study arrhythmogenic diseases. These models have provided important insights into the underlying arrhythmia mechanisms and translational options for their therapeutic management. This position paper from the ESC Working Group on Cardiac Cellular Electrophysiology provides an overview of (i) currently available in vitro, ex vivo, and in vivo electrophysiological research methodologies, (ii) the most commonly used experimental (cellular and animal) models for cardiac arrhythmias including relevant species differences, (iii) the use of human cardiac tissue, induced pluripotent stem cell (hiPSC)-derived and in silico models to study cardiac arrhythmias, and (iv) the availability, relevance, limitations, and opportunities of these cellular and animal models to recapitulate specific acquired and inherited arrhythmogenic diseases, including atrial fibrillation, heart failure, cardiomyopathy, myocarditis, sinus node, and conduction disorders and channelopathies. By promoting a better understanding of these models and their limitations, this position paper aims to improve the quality of basic research in cardiac electrophysiology, with the ultimate goal to facilitate the clinical translation and application of basic electrophysiological research findings on arrhythmia mechanisms and therapies.

Cardiac optical mapping - State-of-the-art and future challenges.

Cardiac optical mapping utilises fluorescent dyes to directly image the electrical function of the heart at a high spatio-temporal resolution which far exceeds electrode techniques. It has therefore become an invaluable tool in cardiac electrophysiological research to map the propagation of heterogeneous electrical signals across the myocardium. In this review, we introduce the principles behind cardiac optical mapping and discuss some of the challenges and state of the art in the field. Key advancements discussed include newly developed fluorescent indicators, tools for the analysis of complex datasets, panoramic imaging systems and technical and computational approaches to realise optical mapping in freely beating hearts.

Artificial Intelligence and Machine Learning in Arrhythmias and Cardiac Electrophysiology.

Artificial intelligence (AI) and machine learning (ML) in medicine are currently areas of intense exploration, showing potential to automate human tasks and even perform tasks beyond human capabilities. Literacy and understanding of AI/ML methods are becoming increasingly important to researchers and clinicians. The first objective of this review is to provide the novice reader with literacy of AI/ML methods and provide a foundation for how one might conduct an ML study. We provide a technical overview of some of the most commonly used terms, techniques, and challenges in AI/ML studies, with reference to recent studies in cardiac electrophysiology to illustrate key points. The second objective of this review is to use examples from recent literature to discuss how AI and ML are changing clinical practice and research in cardiac electrophysiology, with emphasis on disease detection and diagnosis, prediction of patient outcomes, and novel characterization of disease. The final objective is to highlight important considerations and challenges for appropriate validation, adoption, and deployment of AI technologies into clinical practice.

A model for human action potential dynamics in vivo

Computational modeling based on experimental data remains an important component in cardiac electrophysiological research, especially because clinical data such as human action potential (AP) dynamics are scarce or limited by practical or ethical concerns. Such modeling has been used to develop and test a variety of mechanistic hypotheses, with the majority of these studies involving the rate dependence of AP duration (APD) including APD restitution and conduction velocity (CV). However, there is very little information regarding the complex dynamics at the boundary of repolarization (or refractoriness) and reexcitability. Here, we developed a "minimal" ionic model of the human AP, based on in vivo human monophasic AP (MAP) recordings obtained during clinical programmed electrical stimulation (PES) to address the progressive decrease in AP take-off potential (TOP) and associated CV slowing seen during three tightly spaced extrastimuli. Recent voltage-clamp data demonstrating the effect of intracellular calcium on sodium current availability were incorporated and were required to reproduce large (>15 mV) elevations in take-off potential and progressive encroachment. Introducing clinically observed APD gradients into the model enabled us to replicate the dynamic response to PES in patients leading to conduction block and reentry formation for the positive, but not the negative, APD gradient. Finally, we modeled the dynamics of reentry and show that spiral waves follow a meandering trajectory with a period of ~180 ms. We conclude that our model reproduces a variety of electrophysiological behavior including the response to sequential premature stimuli and provides a basis for studies of the initiation of reentry in human ventricular tissue.NEW & NOTEWORTHY This work presents a new model of the action potential of the human which reproduces the complex dynamics during premature stimulation in patients.

A Protocol for Transverse Cardiac Slicing and Optical Mapping in Murine Heart.

Thin living tissue slices have recently emerged as a new tissue model for cardiac electrophysiological research. Slices can be produced from human cardiac tissue, in addition to small and large mammalian hearts, representing a powerful in vitro model system for preclinical and translational heart research. In the present protocol, we describe a detailed mouse heart transverse slicing and optical imaging methodology. The use of this technology for high-throughput optical imaging allows study of electrophysiology of murine hearts in an organotypic pseudo two-dimensional model. The slices are cut at right angles to the long axis of the heart, permitting robust interrogation of transmembrane potential (Vm) and calcium transients (CaT) throughout the entire heart with exceptional regional precision. This approach enables the use of a series of slices prepared from the ventricles to measure Vm and CaT with high temporal and spatial resolution, allowing (i) comparison of successive slices which form a stack representing the original geometry of the heart; (ii) profiling of transmural and regional gradients in Vm and CaT in the ventricle; (iii) characterization of transmural and regional profiles of action potential and CaT alternans under stress (e.g., high frequency pacing or β-adrenergic stimulation) or pathological conditions (e.g., hypertrophy). Thus, the protocol described here provides a powerful platform for innovative research on electrical and calcium handling heterogeneity within the heart. It can be also combined with optogenetic technology to carry out optical stimulation; aiding studies of cellular Vm and CaT in a cell type specific manner.

Effects of Calcium, Magnesium, and Potassium Concentrations on Ventricular Repolarization in Unselected Individuals

BackgroundSubclinical changes on the electrocardiogram are risk factors for cardiovascular mortality. Recognition and knowledge of electrolyte associations in cardiac electrophysiology are based on only in vitro models and observations in patients with severe medical conditions. ObjectivesThis study sought to investigate associations between serum electrolyte concentrations and changes in cardiac electrophysiology in the general population. MethodsSummary results collected from 153,014 individuals (54.4% women; mean age 55.1 ± 12.1 years) from 33 studies (of 5 ancestries) were meta-analyzed. Linear regression analyses examining associations between electrolyte concentrations (mmol/l of calcium, potassium, sodium, and magnesium), and electrocardiographic intervals (RR, QT, QRS, JT, and PR intervals) were performed. The study adjusted for potential confounders and also stratified by ancestry, sex, and use of antihypertensive drugs. ResultsLower calcium was associated with longer QT intervals (−11.5 ms; 99.75% confidence interval [CI]: −13.7 to −9.3) and JT duration, with sex-specific effects. In contrast, higher magnesium was associated with longer QT intervals (7.2 ms; 99.75% CI: 1.3 to 13.1) and JT. Lower potassium was associated with longer QT intervals (−2.8 ms; 99.75% CI: −3.5 to −2.0), JT, QRS, and PR durations, but all potassium associations were driven by use of antihypertensive drugs. No physiologically relevant associations were observed for sodium or RR intervals. ConclusionsThe study identified physiologically relevant associations between electrolytes and electrocardiographic intervals in a large-scale analysis combining cohorts from different settings. The results provide insights for further cardiac electrophysiology research and could potentially influence clinical practice, especially the association between calcium and QT duration, by which calcium levels at the bottom 2% of the population distribution led to clinically relevant QT prolongation by >5 ms.

Shox2 influences mesenchymal stem cell fate in a co-culture model invitro.

Sinoatrial node (SAN) dysfunction is a common cardiovascular problem, and the development of a cell sourced biological pacemaker has been the focus of cardiac electrophysiology research. The aim of biological pacemaker therapy is to produce SAN-like cells, which exhibit spontaneous activity characteristic of the SAN. Short stature homeobox 2 (Shox2) is an early cardiac transcription factor and is crucial in the formation and differentiation of the sinoatrial node (SAN). The present study aimed to improve pacemaker function by overexpression of Shox2 in canine mesenchymal stem cells (cMSCs) to induce a phenotype similar to native pacemaker cells. To achieve this objective, the cMSCs were transfected with lentiviral pLentis-mShox2-red fluorescent protein, and then co-cultured with rat neonatal cardiomyocytes (RNCMs) in vitro for 5–7 days. The feasibility of regulating the differentiation of cMSCs into pacemaker-like cells by Shox2 overexpression was investigated. Reverse transcription-quantitative polymerase chain reaction and western blotting showed that Shox2-transfected cMSCs expressed high levels of T box 3, hyperpolarization-activated cyclic nucleotide-gated cation channel and Connexin 45 genes, which participate in SAN development, and low levels of working myocardium genes, Nkx2.5 and Connexin 43. In addition, Shox2-transfected cMSCs were able to pace RNCMs with a rate faster than the control cells. In conclusion, these data indicate that overexpression of Shox2 in cMSCs can greatly enhance the pacemaker phenotype in a co-culture model in vitro.

LongQt: A cardiac electrophysiology simulation platform

Mathematical modeling has been used for over half a century to advance our understanding of cardiac electrophysiology and arrhythmia mechanisms. Notably, computational studies using mathematical models of the cardiac action potential (AP) have provided important insight into the fundamental nature of cell excitability, mechanisms underlying both acquired and inherited arrhythmia, and potential therapies. Ultimately, an approach that tightly integrates mathematical modeling and experimental techniques has great potential to accelerate discovery. Despite the increasing acceptance of mathematical modeling as a powerful tool in cardiac electrophysiology research, there remain significant barriers to its more widespread use in the field, due in part to the increasing complexity of models and growing need for specialization. To help bridge the gap between experimental and theoretical worlds that stands as a barrier to transformational breakthroughs, we present LongQt, which has the following key features:•Cross-platform, threaded application with accessible graphical user interface.•Facilitates advanced computational cardiac electrophysiology and arrhythmia studies.•Does not require advanced programming skills.

Contribution of health psychology to clinical health settings: An integrative approach to health care

This article begins by discussing the contribution of health psychology to clinical settings, before examining the application of health psychology to a cardiology outpatient clinic from the view of Consultant Cardiologist, and Clinical Lead in cardiac electrophysiology research, Professor Mark O’Neill. Professor O’Neill is also a placement supervisor for MSc Health Psychology students from King’s College London and describes how these placements have affected the cardiology out–patient clinic and patient management.

Effects of PPARγ agonist on heart rate variability and cardiac mitochondrial function in obese-insulin resistant rats

a Cardiac Electrophysiology Research and Training Center, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand b Cardiac Electrophysiology Unit, Department of Physiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand c Center of Excellence in Cardiac Electrophysiology Research, Chiang Mai University, Chiang Mai, Thailand d Department of Oral Biology and Diagnostic Science, Faculty of Dentistry, Chiang Mai University, Chiang Mai, Thailand

Open Source Modeling of Heart Rhythm and Cardiac Pacing

Background: Despite the progress of cardiac arrhythmia research and the advance of the pacemaker technology, much remains unknown about how the external pacing interacts with the heart's intrinsic activity. Methods: We present an open source computer model that is capable to simulate intrinsic heart rhythm under both normal and pathological conditions, and its interactions with extrinsic dual-chamber cardiac pacing. Detailed model structure and the software implementation are described. Results: We demonstrate the usage of the computer model for generating realistic cardiac activation sequences under different rhythm conditions. Representative examples are also provided to illustrate the application of the model to evaluate the performance of pacing algorithms. Conclusions: The present computer model provides a unified platform wherein it is possible to bench test advanced pacemaker algorithms in the presence of different heart rhythms. The availability of this open source model promises to support and stimulate future research in cardiac electrophysiology and development in pacemaker technology.

The Origin and Arrhythmogenic Potential of Fibroblasts in Cardiac Disease

Fibroblasts play a major role in normal cardiac physiology and in the response of the heart to injury and disease. Cardiac electrophysiological research has primarily focused on the mechanisms of remodeling that accompany cardiac disease with an emphasis on myocyte electrophysiology. Recently, there has been increasing interest in the potential role of fibroblasts in cardiac electrophysiology. This review focuses on the arrhythmia mechanisms involving interactions between myocytes and fibroblasts. We also discuss the available evidence supporting the contribution of intracardiac and extracardiac sources to the fibroblast and myofibroblast populations in diseased hearts.

Action Potential Duration Maps in Homogeneous Cardiac Ventricular Tissue Slices are Independent of Changes in Stimulation Site

Cardiac slices are an increasingly popular model system for cardiac electrophysiology research, as they combine ease of handling with patho-physiologically relevant cell-type representation and distribution. The revival of the cardiac slice technique, first established in the 1980's1 has been linked to improved techniques for preparation and monitoring. It now offers a well-posed target for computational modelling of cardiac structure-function interrelations.This study used slices from the New Zealand white rabbits (female, ∼1kg, n=12), cut tangentially to the left ventricular wall surface (350um thick, ∼1x2cm large), offering a simplified ‘pseudo-2D’ experimental model. Slices, loaded with Di-4-ANBDQPQ (voltage sensitive dye; 10uM) to optically monitor action potential duration (APD), were stimulated at four different sites with concentric-bipolar point-electrodes, using a biphasic rectangular pulse 50% above activation threshold and, for comparison, with field stimulation.APD at 50% and 80% repolarization (APD50 and APD80, respectively), was used to establish APD-maps in each slice. Locally-resolved average APD patterns were obtained for pacing frequencies of 1-4Hz. In addition, at each slice location the largest (and smallest) APD-values were selected to create a maximum (and minimum) APD-map. This illustrated the variability-range per slice which, as an example for APD80 at 2Hz, was 196.37ms±6.59ms (maximum; mean±SD), 187.00ms±6.57ms (minimum), and 191.69ms±5.44ms (average). Interestingly, even though the spread of activation differed significantly for the different stimulus sites, resulting APD-maps were comparable for all pacing rates in homogeneous slices (i.e. in the absence of pronounced source-sink mismatches).Thus, APD heterogeneity in cardiac tissue slices shows a reproducible intrinsic pattern that prevails regardless of acute changes in stimulation site and frequency.Claycomb. J Biol Chem. 1976/251:P6082-P6089.