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Cardiac fibroblasts (CFs) play critical roles in heart development, homeostasis, and disease. The limited availability of human CFs from native heart impedes investigations of CF biology and their role in disease. Human pluripotent stem cells (hPSCs) provide a highly renewable and genetically defined cell source, but efficient methods to generate CFs from hPSCs have not been described. Here, we show differentiation of hPSCs using sequential modulation of Wnt and FGF signaling to generate second heart field progenitors that efficiently give rise to hPSC-CFs. The hPSC-CFs resemble native heart CFs in cell morphology, proliferation, gene expression, fibroblast marker expression, production of extracellular matrix and myofibroblast transformation induced by TGFβ1 and angiotensin II. Furthermore, hPSC-CFs exhibit a more embryonic phenotype when compared to fetal and adult primary human CFs. Co-culture of hPSC-CFs with hPSC-derived cardiomyocytes distinctly alters the electrophysiological properties of the cardiomyocytes compared to co-culture with dermal fibroblasts. The hPSC-CFs provide a powerful cell source for research, drug discovery, precision medicine, and therapeutic applications in cardiac regeneration.

Aims: Myocardial infarction (MI) alters cardiac fibre organization with unknown consequences on ventricular arrhythmia. We used diffusion tensor imaging (DTI) of three-dimensional (3D) cardiac fibres and scar reconstructions to identify the main parameters associated with ventricular arrhythmia inducibility and ventricular tachycardia (VT) features after MI., Methods and Results: Twelve pigs with established MI and three controls underwent invasive electrophysiological characterization of ventricular arrhythmia inducibility and VT features. Animal-specific 3D scar and myocardial fibre distribution were obtained from ex vivo high-resolution contrast-enhanced T1 mapping and DTI sequences. Diffusion tensor imaging-derived parameters significantly different between healthy and scarring myocardium, scar volumes, and left ventricular ejection fraction (LVEF) were included for arrhythmia risk stratification and correlation analyses with VT features. Ventricular fibrillation (VF) was the only inducible arrhythmia in 4 out of 12 infarcted pigs and all controls. Ventricular tachycardia was also inducible in the remaining eight pigs during programmed ventricular stimulation. A DTI-based 3D fibre disorganization index (FDI) showed higher disorganization within dense scar regions of VF-only inducible pigs compared with VT inducible animals (FDI: 0.36; 0.36-0.37 vs. 0.32; 0.26-0.33, respectively, P = 0.0485). Ventricular fibrillation induction required lower programmed stimulation aggressiveness in VF-only inducible pigs than VT inducible and control animals. Neither LVEF nor scar volumes differentiated between VF and VT inducible animals. Re-entrant VT circuits were localized within areas of highly disorganized fibres. Moreover, the FDI within heterogeneous scar regions was associated with the median VT cycle length per animal (R2 = 0.5320)., Conclusion: The amount of scar-related cardiac fibre disorganization in DTI sequences is a promising approach for ventricular arrhythmia stratification after MI., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Atrial fibrillation (AF) is a common cardiac arrhythmia and a major risk factor for stroke, heart failure, and premature death. The pathogenesis of AF remains poorly understood, which contributes to the current lack of highly effective treatments. To understand the genetic variation and biology underlying AF, we undertook a genome-wide association study (GWAS) of 6,337 AF individuals and 61,607 AF-free individuals from Norway, including replication in an additional 30,679 AF individuals and 278,895 AF-free individuals. Through genotyping and dense imputation mapping from whole-genome sequencing, we tested almost nine million genetic variants across the genome and identified seven risk loci, including two novel loci. One novel locus (lead single-nucleotide variant [SNV] rs12614435; p = 6.76 × 10 -18 ) comprised intronic and several highly correlated missense variants situated in the I-, A-, and M-bands of titin, which is the largest protein in humans and responsible for the passive elasticity of heart and skeletal muscle. The other novel locus (lead SNV rs56202902; p = 1.54 × 10 -11 ) covered a large, gene-dense chromosome 1 region that has previously been linked to cardiac conduction. Pathway and functional enrichment analyses suggested that many AF-associated genetic variants act through a mechanism of impaired muscle cell differentiation and tissue formation during fetal heart development., (Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Ventricular fibrillation (VF) is the most severe cardiac rhythm disturbance and one of the most important immediate causes of sudden cardiac death. In the structurally normal heart, a small number of stable reentrant sources, perhaps 1 or 2, underlie the mechanism of VF, and the stabilization of the sources, their frequency, and the complexity of the turbulent waves they generate depend on the expression, spatial distribution, and intermolecular interactions of the 2 most important ion channels that control cardiac excitability: the inward rectifier potassium channel, Kir2.1, and the alpha subunit of the main cardiac sodium channel, NaV1.5., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Moderate global cooling of myocardial tissue was shown to destabilize 2-dimensional (2-D) reentry and facilitate its termination., Objective: This study sought to test the hypothesis that regional cooling destabilizes rotors and facilitates termination of spontaneous and DC shock-induced subepicardial reentry in isolated, endocardially ablated rabbit hearts., Methods: Fluorescent action potential signals were recorded from 2-D subepicardial ventricular myocardium of Langendorff-perfused rabbit hearts. Regional cooling (by 5.9°C ± 1.3°C) was applied to the left ventricular anterior wall using a transparent cooling device (10 mm in diameter)., Results: Regional cooling during constant stimulation (2.5 Hz) prolonged the action potential duration (by 36% ± 9%) and slightly reduced conduction velocity (by 4% ± 4%) in the cooled region. Ventricular tachycardias (VTs) induced during regional cooling terminated earlier than those without cooling (control): VTs lasting >30 seconds were reduced from 17 of 39 to 1 of 61. When regional cooling was applied during sustained VTs (>120 seconds), 16 of 33 (48%) sustained VTs self-terminated in 12.5 ± 5.1 seconds. VT termination was the result of rotor destabilization, which was characterized by unpinning, drift toward the periphery of the cooled region, and subsequent collision with boundaries. The DC shock intensity required for cardioversion of the sustained VTs decreased significantly by regional cooling (22.8 ± 4.1 V, n = 16, vs 40.5 ± 17.6 V, n = 21). The major mode of reentry termination by DC shocks was phase resetting in the absence of cooling, whereas it was unpinning in the presence of cooling., Conclusion: Regional cooling facilitates termination of 2-D reentry through unpinning of rotors., (Copyright © 2012 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Cardiac experimental electrophysiology is in need of a well-defined Minimum Information Standard for recording, annotating, and reporting experimental data. As a step towards establishing this, we present a draft standard, called Minimum Information about a Cardiac Electrophysiology Experiment (MICEE). The ultimate goal is to develop a useful tool for cardiac electrophysiologists which facilitates and improves dissemination of the minimum information necessary for reproduction of cardiac electrophysiology research, allowing for easier comparison and utilisation of findings by others. It is hoped that this will enhance the integration of individual results into experimental, computational, and conceptual models. In its present form, this draft is intended for assessment and development by the research community. We invite the reader to join this effort, and, if deemed productive, implement the Minimum Information about a Cardiac Electrophysiology Experiment standard in their own work., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: The intrinsic neural plexus of the mouse heart has not been adequately investigated despite the extensive use of this species in experimental cardiology., Objective: The purpose of this study was to determine the distribution of cholinergic, adrenergic, and sensory neural components in whole-mount mouse heart preparations using double immunohistochemical labeling., Methods/results: Intrinsic neurons were concentrated within 19 ± 3 ganglia (n = 20 mice) of varying size, scattered on the medial side of the inferior caval (caudal) vein on the right atrium and close to the pulmonary veins on the left atrium. Of a total of 1,082 ± 160 neurons, most somata (83%) were choline acetyltransferase (ChAT) immunoreactive, whereas 4% were tyrosine hydroxylase (TH) immunoreactive; 14% of ganglionic cells were biphenotypic for ChAT and TH. The most intense ChAT staining was observed in axonal varicosities. ChAT was evident in nerve fibers interconnecting intrinsic ganglia. Both ChAT and TH immunoreactivity were abundant within the nerves accessing the heart. However, epicardial TH-immunoreactive nerve fibers were predominant on the dorsal and ventral left atrium, whereas most ChAT-positive axons proceeded on the heart base toward the large intrinsic ganglia and on the epicardium of the root of the right cranial vein. Substance P-positive and calcitonin gene-related peptide-immunoreactive nerve fibers were abundant on the epicardium and within ganglia adjacent to the heart hilum. Small intensely fluorescent cells were grouped into clusters of 3 to 8 and were dispersed within large ganglia or separately on the atrial and ventricular walls., Conclusion: Although some nerves and neuronal bundles of the mouse epicardial plexus are mixed, most express either adrenergic or cholinergic markers. Therefore, selective stimulation and/or ablation of the functionally distinct intrinsic neural pathways should allow the study of specific effects on cardiac function., (Copyright © 2011 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Mammalian enabled (Mena) of the Drosophila enabled/vasodilator-stimulated phosphoprotein gene family is a cytoskeletal protein implicated in actin regulation and cell motility. Cardiac Mena expression is enriched in intercalated discs (ICD), the critical intercellular communication nexus between adjacent muscle cells. We previously identified Mena gene expression to be a key predictor of human and murine heart failure (HF). To determine the in vivo function of Mena in the heart, we assessed Mena protein expression in multiple HF models and characterized the effects of genetic Mena deletion on cardiac structure and function. Immunoblot analysis revealed significant upregulation of Mena protein expression in left ventricle tissue from patients with end-stage HF, calsequestrin-overexpressing mice, and isoproterenol-infused mice. Characterization of the baseline cardiac function of adult Mena knockout mice (Mena(-/-)) via echocardiography demonstrated persistent cardiac dysfunction, including a significant reduction in percent fractional shortening compared with wild-type littermates. Electrocardiogram PR and QRS intervals were significantly prolonged in Mena(-/-) mice, manifested by slowed conduction on optical mapping studies. Ultrastructural analysis of Mena(-/-) hearts revealed disrupted organization and widening of ICD structures, mislocalization of the gap junction protein connexin 43 (Cx43) to the lateral borders of cardiomyoycytes, and increased Cx43 expression. Furthermore, the expression of vinculin (an adherens junction protein) was significantly reduced in Mena(-/-) mice. We report for the first time that genetic ablation of Mena results in cardiac dysfunction, highlighted by diminished contractile performance, disrupted ICD structure, and slowed electrical conduction.

Background: Both normal and genetically modified mice are excellent models for investigating molecular mechanisms of arrhythmogenic cardiac diseases that may be associated with an imbalance between sympathetic and parasympathetic nervous input to the heart., Objective: The purpose of this study was to (1) determine the structural organization of the mouse cardiac neural plexus, (2) identify extrinsic neural sources and their relationship with the cardiac plexus, and (3) reveal any anatomic differences in the cardiac plexus between mouse and other species., Methods: Cardiac nerve structures were visualized using histochemical staining for acetylcholinesterase (AChE) on whole heart and thorax-dissected preparations derived from 25 mice. To confirm the reliability of staining parasympathetic and sympathetic neural components in the mouse heart, we applied a histochemical method for AChE and immunohistochemistry for tyrosine hydroxylase (TH) and/or choline acetyltransferase (ChAT) on whole mounts preparations from six mice., Results: Double immunohistochemical labeling of TH and ChAT on AChE-positive neural elements in mouse whole mounts demonstrated equal staining of nerves and ganglia for AChE that were positive for both TH and ChAT. The extrinsic cardiac nerves access the mouse heart at the right and left cranial veins and interblend within the ganglionated nerve plexus of the heart hilum that is persistently localized on the heart base. Nerves and bundles of nerve fibers extend epicardially from this plexus to atria and ventricles by left dorsal, dorsal right atrial, right ventral, and ventral left atrial routes or subplexuses. The right cranial vein receives extrinsic nerves that mainly originate from the right cervicothoracic ganglion and a branch of the right vagus nerve, whereas the left cranial vein is supplied by extrinsic nerves from the left cervicothoracic ganglion and the left vagus nerve. The majority of intrinsic cardiac ganglia are localized on the heart base at the roots of the pulmonary veins. These ganglia are interlinked by interganglionic nerves into the above mentioned nerve plexus of the heart hilum. In general, the examined hearts contained 19 ± 3 ganglia, giving a cumulative ganglion area of 0.4 ± 0.1 mm(2)., Conclusion: Despite substantial anatomic differences in ganglion number and distribution, the structural organization of the intrinsic ganglionated plexus in the mouse heart corresponds in general to that of other mammalian species, including human., (Copyright © 2011 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Atrial and ventricular tachyarrhythmias can be perpetuated by up-regulation of inward rectifier potassium channels. Thus, it may be beneficial to block inward rectifier channels under conditions in which their function becomes arrhythmogenic (e.g., inherited gain-of-function mutation channelopathies, ischemia, and chronic and vagally mediated atrial fibrillation). We hypothesize that the antimalarial quinoline chloroquine exerts potent antiarrhythmic effects by interacting with the cytoplasmic domains of Kir2.1 (I(K1)), Kir3.1 (I(KACh)), or Kir6.2 (I(KATP)) and reducing inward rectifier potassium currents. In isolated hearts of three different mammalian species, intracoronary chloroquine perfusion reduced fibrillatory frequency (atrial or ventricular), and effectively terminated the arrhythmia with resumption of sinus rhythm. In patch-clamp experiments chloroquine blocked I(K1), I(KACh), and I(KATP). Comparative molecular modeling and ligand docking of chloroquine in the intracellular domains of Kir2.1, Kir3.1, and Kir6.2 suggested that chloroquine blocks or reduces potassium flow by interacting with negatively charged amino acids facing the ion permeation vestibule of the channel in question. These results open a novel path toward discovering antiarrhythmic pharmacophores that target specific residues of the cytoplasmic domain of inward rectifier potassium channels.

Background: Sheep are routinely used in experimental cardiac electrophysiology and surgery., Objective: The purpose of this study was to (1) ascertain the topography and architecture of the ovine epicardial neural plexus (ENP), (2) determine the relationships of ENP with vagal and sympathetic cardiac nerves and ganglia, and (3) evaluate gross anatomic differences and similarities of ENP in humans, sheep, and other species., Methods: Ovine ENP and extrinsic sympathetic and vagal nerves were stained histochemically for acetylcholinesterase in whole heart and/or thorax-dissected preparations from 23 newborn lambs, with subsequent examination by stereomicroscope., Results: Intrinsic cardiac nerves extend from the venous part of the ovine heart hilum along the roots of the cranial (superior) caval and left azygos veins to both atria and ventricles via five epicardial routes: dorsal right atrial, middle dorsal, left dorsal, right ventral, and ventral left atrial nerve subplexuses. Intrinsic nerves proceeding from the arterial part of the heart hilum along the roots of the aorta and pulmonary trunk extend exclusively into the ventricles as the right and left coronary subplexuses. The dorsal right atrial, right ventral, and middle dorsal subplexuses receive the main extrinsic neural input from the right cervicothoracic and right thoracic sympathetic T(2) and T(3) ganglia as well as from the right vagal nerve. The left dorsal is supplied by sizeable extrinsic nerves from the left thoracic T(4)-T(6) sympathetic ganglia and the left vagal nerve. Sheep hearts contained an average of 769 +/- 52 epicardial ganglia. Cumulative areas of epicardial ganglia on the root of the cranial vena cava and on the wall of the coronary sinus were the largest of all regions (P <.05)., Conclusion: Despite substantial interindividual variability in the morphology of ovine ENP, right-sided epicardial neural subplexuses supplying the sinoatrial and atrioventricular nodes are mostly concentrated at a fat pad between the right pulmonary veins and the cranial vena cava. This finding is in sharp contrast with a solely left lateral neural input to the human atrioventricular node, which extends mainly from the left dorsal and middle dorsal subplexuses. The abundance of epicardial ganglia distributed widely along the ovine ventricular nerves over respectable distances below the coronary groove implies a distinctive neural control of the ventricles in human and sheep hearts., (Copyright 2010 Heart Rhythm Society. All rights reserved.)

Background: Both atrial stretch and combined adrenocholinergic stimulation (ACS) have been shown to favor initiation and maintenance of atrial fibrillation (AF). Their respective contributions to the electrophysiological mechanism remains, however, incompletely understood., Objective: This study endeavored to determine the mechanism of maintenance of stretch-related AF (SRAF) in the presence and absence of ACS and to assess how focal discharges interact with rotors to modify the level of complexity in the activation patterns to perpetuate AF., Methods: Video imaging of AF dynamics was carried out using a SRAF model in isolated sheep hearts (n = 24). Pharmacological approaches were used to (1) mimic ACS with acetylcholine (1 microM) plus isoproterenol (0.03 microM), and (2) abolish triggered activity, in response to sarcoplasmic reticulum calcium release, with caffeine (5 mM, CA) or ryanodine (10 to 40 microM, RYA)., Results: In the absence of ACS, on perfusion of CA or RYA, focal discharges were abolished and SRAF was terminated in most of the cases (10 of 13 experiments). In the presence of ACS, multiple drifting rotors as well as a large number of focal discharges were identified and only 1 of 11 AF episodes was terminated., Conclusions: In the absence of ACS, SRAF is maintained by high-frequency focal discharges that generate fibrillatory conduction and wave breaks. In the presence of ACS, SRAF dynamics is characterized by multiple high frequency rotors that are rendered unstable by spatially distributed focal discharges.

Background: Spatially discordant alternans (SDA) has been linked to life-threatening arrhythmias. The mechanisms underlying SDA development in cardiac tissue remain unclear., Methods and Results: We investigated the role of conduction velocity (CV) restitution and short-term memory in the organization and evolution of alternans in action potential duration using high-resolution optical mapping of the epicardial surface in 8 isolated, Langendorff-perfused rabbit hearts. To assess the spatial organization of alternans, we tracked the evolution of nodal lines that separate out-of-phase regions of SDA. We measured the action potential duration heterogeneity index and maximal slope of CV restitution and estimated the effects of short-term memory by calculating time constant of action potential duration accommodation (tau). We found that 2 mechanisms underlie the development of SDA in the heart, leading to 2 distinct behaviors of nodal lines. The first mechanism is based on steep CV restitution and is associated with small tau and stable nodal lines. The second mechanism is associated with short-term memory (large tau) and is characterized by shallow CV restitution and unstable behavior of nodal lines. The maximum slope of the CV restitution was steeper (18.16+/-3.34 m/s(2)) and tau was smaller (tau=4.31+/-0.33 stimuli) for areas with stable nodal lines than for areas with unstable nodal lines (6.32+/-0.96 m/s(2) and tau=10.3+/-1.84 stimuli; P<0.01)., Conclusions: Our results provide new insight into the mechanisms underlying SDA formation in the rabbit heart. Specifically, our results suggest that a new mechanism associated with short-term memory underlies SDA formation in the heart, in addition to steep CV restitution.

Recent new information on the dynamics and molecular mechanisms of electrical rotors and spiral waves has increased our understanding of both atrial fibrillation and ventricular fibrillation. In this brief review, we evaluate the available evidence for the separate roles played by individual sarcolemmal ion channels in atrial fibrillation and ventricular fibrillation, assessing the clinical relevance of such findings. Importantly, although human data support the idea that rotors are a crucial mechanism for fibrillation maintenance in both atria and ventricles, there are clear inherent differences between the 2 chamber types, particularly in regard to the role of specific ion channels in fibrillation. But there also are similarities. This knowledge, together with new information on the changes that take place during disease evolution and between structurally normal and diseased hearts, may enhance our understanding of fibrillatory processes pointing to new approaches to improve disease outcomes.

Changes in ventricular fibrillation (VF) organization occurring after the onset of global ischemia are relevant to defibrillation and survival but remain poorly understood. We hypothesized that ischemia-specific dynamic instability of the action potential (AP) causes a loss of spatiotemporal periodicity of propagation and broadening of the electrocardiogram (ECG) frequency spectrum during VF in the ischemic myocardium. We recorded voltage-sensitive fluorescence of di-4-ANEPPS (anterior left ventricle, 35 x 35 mm, 64 x 64 pixels) and the volume-conducted ECG in six blood-perfused hearts during 10 min of VF and global ischemia. We used coefficient of variation (CV) to estimate variability of AP amplitude, AP duration, and diastolic interval (CV-APA, CV-APD, and CV-DI, respectively). We computed excitation median frequency (Median&#95;F), spectral width of the AP and ECG (SpW-AP and SpW-ECG, respectively), wavebreak incidence (WBI), and recurrence of propagation direction (RPD). We found three distinct phases of local VF dynamics: "relatively periodic" (

Previous studies have suggested an important role for the inward rectifier K+ current (I K1) in stabilizing rotors responsible for ventricular tachycardia (VT) and fibrillation (VF). To test this hypothesis, we used a line of transgenic mice (TG) overexpressing Kir 2.1-green fluorescent protein (GFP) fusion protein in a cardiac-specific manner. Optical mapping of the epicardial surface in ventricles showed that the Langendorff-perfused TG hearts were able to sustain stable VT/VF for 350 +/- 1181 s at a very high dominant frequency (DF) of 44.6 +/- 4.3 Hz. In contrast, tachyarrhythmias in wild-type hearts (WT) were short-lived (3 +/- 9 s), and the DF was 26.3 +/- 5.2 Hz. The stable, high frequency, reentrant activity in TG hearts slowed down, and eventually terminated in the presence of 10 mum Ba2+, suggesting an important role for I K1. Moreover, by increasing I K1 density in a two-dimensional computer model having realistic mouse ionic and action potential properties, a highly stable, fast rotor (approximately 45 Hz) could be induced. Simulations suggested that the TG hearts allowed such a fast and stable rotor because of both greater outward conductance at the core and shortened action potential duration in the core vicinity, as well as increased excitability, in part due to faster recovery of Na+ current. The latter resulted in a larger rate of increase in the local conduction velocity as a function of the distance from the core in TG compared to WT hearts, in both simulations and experiments. Finally, simulations showed that rotor frequencies were more sensitive to changes (doubling) in I K1, compared to other K+ currents. In combination, these results provide the first direct evidence that I K1 up-regulation in the mouse heart is a substrate for stable and very fast rotors.

The long QT syndrome type-3 (LQT3) is an inherited cardiac disorder caused by mutations in the sodium channel gene SCN5A. LQT3 has been associated with ventricular arrhythmias and sudden cardiac death, specially at low heart rates. Based on computer simulations and experimental investigations, analysis of the morphology of the Action Potential (AP) has shown that it undergoes early afterdepolarizations (EADs) and spontaneous discharges, which are thought to be the trigger for reentry like-activity. However, dynamic characteristics of cardiac tissue are also important factors of arrhythmia mechanisms. In this work, we propose a dynamical analysis of the LQT3 at cellular level. We use a detailed Markovian model of the DeltaKPQ mutation, which is associated with LQT3, and we study beat-to-beat AP Duration (APD) variations by using a long-term stimulation protocol. Compared to wild-type (WT) cells, DeltaKPQ mutant cells are found to develop APD alternans over a narrow range of stimulation frequencies. Moreover, the interval of frequency dependence of APD alternans is related to the degree of severity of the EADs present in the AP. In conclusion, dynamical analysis of paced cells is a useful approach to understand the mechanisms of rate dependent arrhythmias.

Objectives: The purpose of this study was to investigate scaling of the duration of late diastolic left ventricular (LV) filling in relation to AV conduction time (delay) (PR interval on the ECG) in mammals., Background: From mouse to whale, AV delay increases 10-fold, whereas body mass increases one million-fold. The apparent "mismatch" results from scaling of AV delay versus body and heart mass., Methods: We measured (1) mitral orifice diameter in 138 postmortem hearts of 48 mammalian species weighing between 17 g and 250 kg and (2) transmitral diastolic flow using magnetic resonance imaging (MRI) recordings of 10 healthy human individuals. (3) We visually inspected early and late diastolic LV filling. (4) We developed two physical models to explain scaling of late diastolic LV filling time., Results: (1) Diameter of the mitral orifice proportionally relates to heart length (third root of heart mass). (2) Atrial contraction starts at a fixed instant (+/- 80%) of the (normalized) cardiac cycle and contributes 31% +/- 5% to LV filling. (3) MRI shows that during diastole, the left atrium (LA) and LV form a single space. (4) The physical models relate the duration of late diastolic LV filling directly to heart length, the third root of heart mass., Conclusions: (1) Late diastolic (LV) filling time scales with heart length (third root of heart mass). (2) No "mismatch" exists between AV delay and heart size. (3) Knowledge of the actual starting time of atrial contraction may contribute to better treatment of patients with heart failure. (4) The findings suggest that in evolution of mammalian species, hemodynamics commands electrical behavior of the heart.

Voltage-sensitive fluorescent dyes are commonly used to measure cardiac electrical activity. Recent studies indicate, however, that optical action potentials (OAPs) recorded from the myocardial surface originate from a widely distributed volume beneath the surface and may contain useful information regarding intramural activation. The first step toward obtaining this information is to predict OAPs from known patterns of three-dimensional (3-D) electrical activity. To achieve this goal, we developed a two-stage model in which the output of a 3-D ionic model of electrical excitation serves as the input to an optical model of light scattering and absorption inside heart tissue. The two-stage model permits unique optical signatures to be obtained for given 3-D patterns of electrical activity for direct comparison with experimental data, thus yielding information about intramural electrical activity. To illustrate applications of the model, we simulated surface fluorescence signals produced by 3-D electrical activity during epicardial and endocardial pacing. We discovered that OAP upstroke morphology was highly sensitive to the transmural component of wave front velocity and could be used to predict wave front orientation with respect to the surface. These findings demonstrate the potential of the model for obtaining useful 3-D information about intramural propagation.

Three-dimensional rotors, or scroll waves, provide essential insight into the activity of excitable media. They also are a suspected cause in the formation and maintenance of ventricular fibrillation, whose lethality is well known. It is therefore of considerable interest to find out what configurations can be adopted by such pathologies. A scroll's behavior is embodied in its organizing center or filament, a largely quiescent tube about which the scroll rotates. Predicting filament shape has normally required computer-intensive simulations of the whole scroll in time. We have found a fast and robust principle that yields the prediction for stationary filaments on a purely geometrical basis, blind to the reaction parameters of the medium. The procedure is to calculate the filament shape as a minimal path. We work in singly diffusive media whose diffusivity tensor--and no other feature--varies spatially. Mathematical and numerical evidence is presented for the proposition that a stable filament is a geodesic in a three-dimensional space whose metric is given by the inverse diffusivity tensor of the medium. Away from the boundaries, a stable filament is unaffected by the reaction parameters. The algorithmic aspects of this work are subsidiary to our main purpose of drawing attention to the universal and unexpectedly exact fit of an elementary geodesic principle within reaction-diffusion theories.

We studied the effect of sinusoidal electric fields on cardiac tissue both experimentally and numerically. We found that periodic forcing at 5-20 Hz using voltage applied in the bathing solution could stop the propagation of excitation waves by producing standing waves of membrane depolarization. These patterns were independent of the driving frequency in contrast to classical standing waves. The stimulus strength required for pattern formation was large compared to the excitation threshold. A novel tridomain representation of cardiac tissue was required to reproduce this behavior numerically.

Studies have characterized conduction velocity in the right and left bundle branches (RBB, LBB) of normal and genetically engineered mice. However, no information is available on the action potential characteristics of the specialized conduction system (SCS). We have used microelectrode techniques to characterize action potential properties of the murine SCS, as well as epicardial and endocardial muscle preparations for comparison. In the RBB, action potential duration at 50%, 70%, and 90% repolarization (APD(50,70,90)) was 6+/-0.7, 35+/-6, and 90+/-7 ms, respectively. Maximum upstroke velocity (dV/dt(max)) was 153+/-14 V/s, and conduction velocity averaged 0.85+/-0.2 m/s. APD(90) was longer in the Purkinje network of fibers (web) than in the RBB (P<0.01). Web APD(50) was longer in the left than in the right ventricle (P<0.05). Yet, web APD(90) was longer in the right than in the left ventricle (P<0.001). APD(50,70) was significantly longer in the endocardial than in the epicardial (P<0.001; P<0.003). APD(90) in the epicardial and endocardial was shorter than in the RBB ( approximately 36 ms versus approximately 100 ms). Spontaneous electrical oscillations in phase 2 of the SCS occasionally resulted in early afterdepolarizations. These results demonstrate that APDs in the murine SCS are significantly ( approximately 2-fold) longer than in the myocardium and implicate the role of the murine SCS in arrhythmias. The differences should have important implications in the use of the mouse heart to study excitation, propagation, and arrhythmias.

Scroll waves of electrical excitation in heart tissue are implicated in the development of lethal cardiac arrhythmias. Here we study the relation between the geometry of myocardial fibers and the equilibrium shape of a scroll wave filament. Our theory accommodates a wide class of myocardial models with spatially varying diffusivity tensor, adjusted to fit fiber geometry. We analytically predict the exact equilibrium shapes of the filaments. The major conclusion is that the filament shape is a compromise between a straight line and full alignment with the fibers. The degree of alignment increases with the anisotropy ratio. The results, being purely geometrical, are independent of details of ionic membrane mechanisms. Our theoretical predictions have been verified to excellent accuracy by numerically simulating the stable equilibration of a scroll filament in a model of the FitzHugh-Nagumo type.

Introduction: Atrial fibrillation (AF) is characterized by complex wave propagation, yet periodic excitation suggesting a high degree of organization may be revealed during sustained AF. We provide a systematic quantification of the spatial distribution of dominant frequencies (DFs) of local excitation on the epicardium of the right atrial (RA) free wall and left atrial (LA) appendage of the isolated sheep heart during AF. The data reveal, for the first time, hidden organization, independent of the activation sequences or nature of electrograms., Methods and Results: In 13 Langendorff-perfused sheep hearts, AF was induced in presence of 0.1 to 0.6 microM acetylcholine. Video movies (potentiometric dye di-4-ANEPPS) of the RA and LA (>30,000 and >20,000 pixels, respectively) were obtained at 120 frames/sec and a biatrial electrogram was recorded. Spectral analyses were performed on movies with DF maps constructed. During AF, the activity formed stable discrete domains with uniform DFs within each domain. Acceleration of AF increased the number of domains (R = 0.81, P < 0.0001) and the DF variance (R = 0.63, P < 0.001), indicating a decrease in organization. Also, the LA was faster and more homogeneous, with smaller number of DF domains, compared to the RA (P < 0.00001)., Conclusion: In this model, AF is characterized by multiple domains with distinct DFs on the atrial epicardium. The decrease in domain area with increased rate suggests that AF results from high-frequency impulses that undergo spectral transformations. The LA is generally faster and more organized than the RA, suggesting that the sources for the impulses are localized to the LA.

Background: Atrial fibrillation (AF) has traditionally been described as aperiodic or random. Yet, ongoing sources of high-frequency periodic activity have recently been suggested to underlie AF in the sheep heart. Our objective was to use a combination of optical and bipolar electrode recordings to identify sites of periodic activity during AF and elucidate their mechanism., Methods and Results: AF was induced by rapid pacing in the presence of 0.1 to 0.5 micromol/L acetylcholine in 7 Langendorff-perfused sheep hearts. We used simultaneous optical mapping of the right and left atria (RA and LA) and frequency sampling of optical and bipolar electrode recordings (including a roving electrode) to identify sites having the highest dominant frequency (DF). Rotors were identified from optical recordings, and their rotation period, core area, and perimeter were measured. In all, 35 AF episodes were analyzed. Mean LA and RA DFs were 14.7+/-3.8 and 10.3+/-2.1 Hz, respectively. Spatiotemporal periodicity was seen in the LA during all episodes. In 5 of 7 experiments, a single site having periodic activity at the highest DF was localized. The highest DF was most often (80%) localized to the posterior LA, near or at the pulmonary vein ostium. Rotors (n=14) were localized on the LA. The mean core perimeter and area were 10.4+/-2.8 mm and 3.8+/-2.8 mm(2), respectively., Conclusions: Frequency sampling allows rapid identification of discrete sites of high-frequency periodic activity during AF. Stable microreentrant sources are the most likely underlying mechanism of AF in this model.

The mechanism(s) underlying ventricular fibrillation (VF) remain unclear. We hypothesized that at least some forms of VF are not random and that high-frequency periodic sources of activity manifest themselves as spatiotemporal periodicities, which drive VF. Twenty-four VF episodes from 8 Langendorff-perfused rabbit hearts were studied using high-resolution video imaging in conjunction with ECG recordings and spectral analysis. Sequential wavefronts that activated the ventricles in a spatially and temporally periodic fashion were identified. In addition, we analyzed the lifespan and dynamics of wavelets in VF, using a new method of phase mapping that enables identification of phase singularity points (PSs), which flank individual wavelets. Spatiotemporal periodicity was found in 21 of 24 episodes. Complete reentry on the epicardial surface was observed in 3 of 24 episodes. The cycle length of discrete regions of spatiotemporal periodicity correlated highly with the dominant frequency of the optical pseudo-ECG (R(2)=0.75) and with the global bipolar electrogram (R(2)=0.79). The lifespan of PSs was short (14.7+/-14.4 ms); 98% of PSs existed for <1 rotation. The mean number of waves entering (6.50+/-0.69) exceeded the mean number of waves that exited our mapping field (4.25+/-0.56; P<0.05). These results strongly suggest that ongoing stable sources are responsible for the majority of the frequency content of VF and therefore play a role in its maintenance. In this model, multiple wavelets resulting from wavebreaks do not appear to be responsible for the sustenance of this arrhythmia, but are rather the consequence of breakup of high-frequency activation from a dominant reentrant source.

Cell loss contributes to the pathogenesis of many inherited and acquired human diseases. We have developed a system to conditionally ablate cells of any lineage and developmental stage in the mouse by regulated expression of the diphtheria toxin A (DTA) gene by using tetracycline-responsive promoters. As an example of this approach, we targeted expression of DTA to the hearts of adult mice to model structural abnormalities commonly observed in human cardiomyopathies. Induction of DTA expression resulted in cell loss, fibrosis, and chamber dilatation. As in many human cardiomyopathies, transgenic mice developed spontaneous arrhythmias in vivo, and programmed electrical stimulation of isolated-perfused transgenic hearts demonstrated a strikingly high incidence of spontaneous and inducible ventricular tachycardia. Affected mice showed marked perturbations of cardiac gap junction channel expression and localization, including a subset with disorganized epicardial activation patterns as revealed by optical action potential mapping. These studies provide important insights into mechanisms of arrhythmogenesis and suggest that conditional lineage ablation may have wide applicability for studies of disease pathogenesis.

Previous experimental studies have clearly demonstrated the existence of drifting and stationary electrical spiral waves in cardiac muscle and their involvement in cardiac arrhythmias. Here we present results of a study of reentrant excitation in computer simulations based on a membrane model of the ventricular cell. We have explored in detail the parameter space of the model, using tools derived from previous numerical studies in excitation-dynamics models. We have found appropriate parametric conditions for sustained stable spiral wave dynamics (1 s of activity or approximately 10 rotations) in simulations of an anisotropic (ratio in velocity 4:1) cardiac sheet of 2 cm x 2 cm. Initially, we used a model that reproduced well the characteristics of planar electrical waves exhibited by thin sheets of sheep ventricular epicardial muscle during rapid pacing at a cycle length of 300 ms. Under these conditions, the refractory period was 147 ms; the action potential duration (APD) was 120 ms; the propagation velocity along fibers was 33 cm/s; and the wavelength along fibers was 4.85 cm. Using cross-field stimulation in this model, we obtained a stable self-sustaining spiral wave rotating around an unexcited core of 1.75 mm x 7 mm at a period of 115 ms, which reproduced well the experimental results. Thus the data demonstrate that stable spiral wave activity can occur in small cardiac sheets whose wavelength during planar wave excitation in the longitudinal direction is larger than the size of the sheet. Analysis of the mechanism of this observation demonstrates that, during rotating activity, the core exerts a strong electrotonic influence that effectively abbreviates APD (and thus wavelength) in its immediate surroundings and is responsible for the stabilization and perpetuation of the activity. We conclude that appropriate adjustments in the kinetics of the activation front (i.e., threshold for activation and upstroke velocity of the initiating beat) of currently available models of the cardiac cell allow accurate reproduction of experimentally observed self-sustaining spiral wave activity. As such, the results set the stage for an understanding of functional reentry in terms of ionic mechanisms.

Objectives: This study sought to 1) test the hypothesis that in the setting of bradycardia and drug-induced action potential prolongation, multiple foci of early afterdepolarizations (EADs) result in beat to beat changes in the origin and direction of the excitation wave front and are responsible for polymorphic arrhythmias; and 2) determine whether EADs may initiate nonstationary reentry, giving rise to the typical torsade de pointes (TDP) pattern., Background: In the past, it has been difficult to associate EADs or reentry with the undulating electrocardiographic (ECG) patterns of TDP., Methods: A voltage-sensitive dye was used for high resolution video imaging of electrical waves on the epicardial and endocardial surface of the Langendorff-perfused rabbit heart. ECG and monophasic action potentials from the right septal region were also recorded. Bradycardia was induced by ablation of the atrioventricular node., Results: Perfusion of low potassium chloride Tyrode solution plus quinidine led to prolongation of the action potential and the QT interval. Eventually, EADs and triggered activity ensued, giving rise to intermittent episodes of polymorphic arrhythmia. In one experiment, triggered activity was followed by a long episode of vortex-like reentry with an ECG pattern characteristic of TDP. However, in most experiments, focal activity of varying origins and propagation patterns was observed. Triggered responses also showed varying degrees of local block. Similar results were obtained with E-4031. Burst pacing both at control conditions and in the presence of quinidine consistently led to vortex-like reentry whose ECG pattern resembled TDP. However, the cycle length of the arrhythmia with quinidine was longer than that for control ([mean +/- SEM] 194 +/- 12 vs. 132 +/- 8 ms, p < 0.03)., Conclusions: Drug-induced polymorphic ventricular arrhythmias may result from beat to beat changes in wave propagation patterns initiated by EADs or EAD-induced nonstationary reentrant activity. In contrast, burst pacing-induced polymorphic tachycardia in the presence or absence of drugs is the result of nonstationary reentrant activity.

Background: The effects of defibrillatory shocks on the organization of atrial fibrillation (AF) have not been studied previously. In this study, we examined the events that precede, accompany, and follow the application of atrial defibrillatory shocks., Methods and Results: We used video imaging to study the sequence of activation on the surface of the atria in the Langendorff-perfused sheep heart. We recorded transmembrane potentials simultaneously from > 20000 sites on the epicardium during AF as well as during and after biphasic shocks applied by a programmable atrial defibrillator. Defibrillatory shocks (1.2 +/- 0.6 J; n = 6) depolarized all epicardial regions of the atria, and asynchronous repolarization occurred. The shocks resulted in four types of responses: (1) immediate cessation of epicardial activity. (2) single postshock activation. (3) organized activation for 0.8 to 1.5 seconds followed by termination, and (4) organized activity followed by degeneration back into AF. Types 2 through 4 involved a quiescent period lasting 110 +/- 28 ms immediately after the shock, then an activation sequence similar to those observed during sinus rhythm. The first cycle length after the shock for types 3 and 4 (170 +/- 36 ms) was longer than during AF (144 +/- 33 ms). Repolarization time after a shock was significantly longer for successful compared with unsuccessful shocks., Conclusions: These results indicate that the shock depolarized the entire atrial epicardial surface, followed by a quiescent period, after which organized activation emanated from the sinoatrial pacemaker region.

Ventricular fibrillation is the most important cause of cardiac electrical instability leading to sudden death. Fibrillation is believed to be associated with complex three-dimensional (3-D) spatio-temporal patterns of electrical excitation of the myocardium. However, to this date, such patterns have not been directly observed or characterized, and little is known about their dynamics. In this paper, we present results that strongly support the hypothesis that at least some cases of ventricular fibrillation in the structurally normal heart may be the result of a single 3-D electrical scroll wave that moves rapidly throughout the ventricles, giving rise to complex patterns of cardiac muscle excitation.

In cardiac tissue, during partial blockade of the membrane sodium channels, or at high frequencies of excitation, inexcitable obstacles with sharp edges may destabilize the propagation of electrical excitation waves, causing the formation of self-sustained vortices and turbulent cardiac electrical activity. The formation of such vortices, which visually resembles vortex shedding in hydrodynamic turbulent flows, was observed in sheep epicardial tissue using voltage-sensitive dyes in combination with video-imaging techniques. Vortex shedding is a potential mechanism leading to the spontaneous initiation of uncontrolled high-frequency excitation of the heart.

Background: Ventricular tachycardia may result from vortexlike reentrant excitation of the myocardium. Our general hypothesis is that in the structurally normal heart, these arrhythmias are the result of one or two nonstationary three-dimensional electrical scroll waves activating the heart muscle at very high frequencies., Methods and Results: We used a combination of high-resolution video imaging, electrocardiography, and image processing in the isolated rabbit heart, together with mathematical modeling. We characterized the dynamics of changes in transmembrane potential patterns on the epicardial surface of the ventricles using optical mapping. Image processing techniques were used to identify the surface manifestation of the reentrant organizing centers, and the location of these centers was used to determine the movement of the reentrant pathway. We also used numerical simulations incorporating Fitzhugh-Nagumo kinetics and realistic heart geometry to study how stationary and nonstationary scroll waves are manifest on the epicardial surface and in the simulated ECG. We present epicardial surface manifestations (reentrant spiral waves) and ECG patterns of nonstationary reentrant activity that are consistent with those generated by scroll waves established at the right and left ventricles. We identified the organizing centers of the reentrant circuits on the epicardial surface during polymorphic tachycardia, and these centers moved during the episodes. In addition, the arrhythmias that showed the greatest movement of the reentrant centers displayed the largest changes in QRS morphology. The numerical simulations showed that stationary scroll waves give rise to monomorphic ECG signals, but nonstationary meandering scroll waves give rise to undulating ECGs characteristic of torsade de pointes., Conclusions: Polymorphic ventricular tachycardia in the healthy, isolated rabbit heart is the result of either a single or paired ("figure-of-eight") nonstationary scroll waves. The extent of the scroll wave movement corresponds to the degree of polymorphism in the ECG. These results are consistent with our numerical simulations that showed monomorphic ECG patterns of activity for stationary scroll waves but polymorphic patterns for scroll waves that were nonstationary.

A model study was carried out to investigate the mechanism of changes in excitability at long cycle lengths (i.e., > 1,000 ms), which are responsible for various phenomena, including electrotonic inhibition, active facilitation, and hysteresis of excitability in ventricular muscle at slow frequencies of stimulation. Experimental studies suggested that with repetitive activity the inward rectifier potassium current (IK1) is not a passive component of membrane response and that the dynamics of IK1 are responsible for the changes in excitability at long cycle lengths. In the present study, we have used new experimental data as the basis to modify the equations for IK1 in the ionic model for ventricular muscle of the Luo and Rudy (LR) model. The modified equations for IK1 incorporate an additional slow gate (s-gate), which governs the transition from a high steady-state conductance at rest to a lower conductance with repetitive stimulation. In simulation studies, electronic inhibition was seen in the original and the modified LR model and was shown to depend on changes in the delayed rectifier current (IK). However, addition of the s-gate to IK1 of the LR model extended the frequency dependence of excitability to longer cycle lengths and allowed for the demonstration of active facilitation and hysteresis. These results support the hypothesis that the inward rectifier is involved in the dynamic control of membrane excitability. The overall results provide mechanistic explanations for heart rate-dependent excitation abnormalities that may be involved in the genesis of cardiac arrhythmias.

We have investigated the role of wave-front curvature on propagation by following the wave front that was diffracted through a narrow isthmus created in a two-dimensional ionic model (Luo-Rudy) of ventricular muscle and in a thin (0.5-mm) sheet of sheep ventricular epicardial muscle. The electrical activity in the experimental preparations was imaged by using a high-resolution video camera that monitored the changes in fluorescence of the potentiometric dye di-4-ANEPPS on the surface of the tissue. Isthmuses were created both parallel and perpendicular to the fiber orientation. In both numerical and biological experiments, when a planar wave front reached the isthmus, it was diffracted to an elliptical wave front whose pronounced curvature was very similar to that of a wave front initiated by point stimulation. In addition, the velocity of propagation was reduced in relation to that of the original planar wave. Furthermore, as shown by the numerical results, wave-front curvature changed as a function of the distance from the isthmus. Such changes in local curvature were accompanied by corresponding changes in velocity of propagation. In the model, the critical isthmus width was 200 microns for longitudinal propagation and 600 microns for transverse propagation of a single planar wave initiated proximal to the isthmus. In the experiments, propagation depended on the width of the isthmus for a fixed stimulation frequency. Propagation through an isthmus of fixed width was rate dependent both along and across fibers. Thus, the critical isthmus width for propagation was estimated in both directions for different frequencies of stimulation. In the longitudinal direction, for cycle lengths between 200 and 500 milliseconds, the critical width was < 1 mm; for 150 milliseconds, it was estimated to be between 1.3 and 2 mm; and for the maximum frequency of stimulation (117 +/- 15 milliseconds), it was > 2.5 mm. In the transverse direction, critical width was between 1.78 and 2.32 mm for a basic cycle length of 200 milliseconds. It increased to values between 2.46 and 3.53 mm for a basic cycle length of 150 milliseconds. The overall results demonstrate that the curvature of the wave front plays an important role in propagation in two-dimensional cardiac muscle and that changes in curvature may cause slow conduction or block.

Objective: Diacetyl monoxime (DAM), a nucleophilic agent with "phosphatase-like" activity, has been found to effectively and reversibly block cardiac muscle contraction, while the cells remain capable of generating transmembrane action potentials. The aim of this study was to characterise the effects of DAM on the electrical properties of cardiac muscle., Methods: Sheep epicardial muscle, guinea pig papillary muscle, and guinea pig ventricular myocytes were studied using conventional microelectrode techniques as well as single electrode current and voltage clamp techniques., Results: DAM (5-20 mM) decreased action potential duration at 50% and 90% repolarisation levels (APD50, APD90) and refractory period in a dose dependent manner without causing significant changes in action potential amplitude, maximum upstroke velocity, or resting membrane potential. DAM induced a slight decrease in action potential conduction velocity in both the longitudinal and transverse directions, but on average the conduction velocity recorded in the presence of the drug was not significantly different from control. The time course of the APD restitution curve was not significantly changed but the frequency dependent APD variations were reduced. The ionic bases for these changes were studied in guinea pig ventricular myocytes. As with the results obtained in tissue preparations, DAM 15 mM decreased APD50 and APD90 by 35% and 29%, respectively. Under voltage clamp conditions, DAM led to a 35% reduction of ICa. The delayed rectifier IK current and the inward rectifier background current were also partially depressed by DAM but to a lesser extent. All of these effects were reversible upon washout., Conclusions: Aside from its well known effect as an electromechanical uncoupler, DAM causes a small, reversible, and non-selective reduction of several membrane conductances. Provided such effects are taken into consideration, DAM is a valuable tool in electrophysiological studies.

Excitable media can support spiral waves rotating around an organizing centre. Spiral waves have been discovered in different types of autocatalytic chemical reactions and in biological systems. The so-called 're-entrant excitation' of myocardial cells, causing the most dangerous cardiac arrhythmias, including ventricular tachycardia and fibrillation, could be the result of spiral waves. Here we use a potentiometric dye in combination with CCD (charge-coupled device) imaging technology to demonstrate spiral waves in the heart muscle. The spirals were elongated and the rotation period, Ts, was about 180 ms (3-5 times faster than normal heart rate). In most episodes, the spiral was anchored to small arteries or bands of connective tissue, and gave rise to stationary rotations. In some cases, the core drifted away from its site of origin and dissipated at a tissue border. Drift was associated with a Doppler shift in the local excitation period, T, with T ahead of the core being about 20% shorter than T behind the core.

Subthreshold potentials are thought to be mediated by time-independent, "passive" background currents. In this study, we show that the background current-voltage (I-V) relation of guinea pig ventricular myocytes is changed significantly by repetitive stimulation, in such a way that cell excitability becomes enhanced. Myocytes were used for whole-cell voltage-clamp experiments. A voltage-clamp ramp (100 mV/sec) to -50 mV was applied from a holding potential of -100 mV. Subsequently, a train of square voltage-clamp pulses to +10 mV (duration, 300 msec; interpulse interval, 300 msec) was delivered from a holding potential of -85 mV. A new ramp was applied again immediately after the train, and the resulting I-V curve was compared with that obtained before the train. Pulsing displaced the I-V relation to the right, the zero-current point becoming 1-2 mV less negative, and increased the degree of inward-going rectification. These changes were insensitive to tetrodotoxin (30 microM); disappeared during superfusion with cobalt (2 mM), verapamil (22 microM), or ryanodine (5 microM); and could not be mimicked by agonists of the protein kinase C system. In the presence of cesium (8 mM), pulsing still displaced the I-V curve to the right. However, the linear portion of the curve became steeper after the train. Subtraction of the cesium-sensitive current from control revealed that, although the zero-current point remained constant, the I-V relation showed a stronger inward-going rectification after pulsing. In accordance with these results, we have demonstrated hysteresis of excitability in ventricular myocytes. We conclude that the observed changes are mediated by an increase in intracellular calcium, which leads to an increase in rectification of IK1, as well as to activation of another membrane-conductance system, perhaps the Na-Ca exchange or the Ca(2+)-activated, nonselective current.

Recent studies in isolated cardiac tissue preparations have demonstrated the applicability of a one-dimensional difference equation model describing the global behavior of a driven nonpacemaker cell to the understanding of rate-dependent cardiac excitation. As a first approximation to providing an ionic basis to complex excitation patterns in cardiac cells, we have compared the predictions of the one-dimensional model with those of numerical simulations using a modified high-dimensional ionic model of the space-clamped myocyte. Stimulus-response ratios were recorded at various stimulus magnitudes, durations, and frequencies. Iteration of the difference equation model reproduced all important features of the ionic model results, including a wide spectrum of stimulus-response locking patterns, period doubling, and irregular (chaotic) dynamics. In addition, in the parameter plane, both models predict that the bifurcation structure of the cardiac cell must change as a function of stimulus duration, because stimulus duration modifies the type of supernormal excitability present at short diastolic interval. We conclude that, to a large extent, the bifurcation structure of the ionic model under repetitive stimulation can be understood by two functions: excitability and action potential duration. The characteristics of these functions depend on the stimulus duration.

Chaos is a term used to characterize aperiodic activity arising in a dynamical system, or in a set of equations describing the system's temporal evolution as a result of a deterministic mechanism that has sensitive dependence on initial conditions. Chaos, in that sense, has been proposed to make an important contribution to normal and abnormal cardiac rhythms. To date, however, descriptions of chaos in heart tissue have been limited primarily to periodically forced cardiac pacemakers. Because many cardiac rhythm disturbances, particularly those initiated or perpetuated by re-entrant excitation, originate from within non-pacemaker cardiac tissues, demonstrations of chaos in non-pacemaker tissue might provide a deterministic explanation for a wide variety of complex dysrhythmias. Here we report experimental evidence for chaotic patterns of activation and action potential characteristics in externally driven, non-spontaneously active Purkinje fibres and ventricular muscle. The results indicate that there is an apparent link between the mechanism of low dimensional chaos and the occurrence of reflected responses which could lead to more spatially disorganized phenomena. A detailed mechanism for the low dimensional chaos observed experimentally is pursued using a difference equation model. Critical features of the model include a non-monotonic relationship between recovery time during rhythmic stimulation and the state of membrane properties, and a steeply sloped recovery of membrane properties over certain ranges of recovery times. Besides explaining our results, the analytical model may pertain to irregular dynamics in other excitable systems, particularly the intact dysrhythmic heart.

Excitation and impulse propagation in cardiac tissues are dependent on the heart rate and can occur in extremely complex patterns. In this chapter we present the results of Purkinje fiber experiments and of computer simulations using an ionic (Beeler & Reuter) model for the ventricular cell. We have studied the global rate-dependent behavior of cardiac cells through a systematic analysis of their response to single as well as repetitive depolarizing stimuli, and determined the role of nonlinearity in the mechanism(s) of their behaviors. To this end, we devised an analytical difference equation model of cardiac cell excitation which could be used to predict simple as well as chaotic behavior of both the Purkinje fiber and the Beeler & Reuter cell, depending on the stimulation rate. Both experimental and modeling results suggest that the presence of supernormal recovery in cell excitability establishes sufficient nonlinearity so that, during repetitive stimulation, the dynamics of cell response may be regular and predictable when the stimulus magnitude is either very small or very large, or they may be chaotic and very unpredictable when the stimulus magnitude is intermediate. The overall results suggest that the application of nonlinear systems theory to electrophysiology may have importance in the understanding of cardiac rhythm and conduction disturbances, and may have clinical implications as well.

The ionic mechanisms of slow recovery of cardiac excitability and rate-dependent activation failure were studied in single, enzymatically dissociated guinea pig ventricular myocytes and in computer simulations using a modified version of the Beeler and Reuter model for the ventricular cell. On the basis of our results, we developed a simplified analytical model for recovery of cell excitability during diastole. This model was based on the equations for current distribution in a resistive-capacitive circuit. A critical assumption in the model is that, in the voltage domain of the subthreshold responses, the sodium and calcium inward currents do not play a significant role, and only the two potassium outward currents, the delayed rectifier (IK) and the inward rectifier, are operative. The appropriate parameters needed to numerically solve the analytical model were measured in the guinea pig ventricular myocyte, as well as in the Beeler and Reuter cell. The curves of recovery of excitability and the rate-dependent activation patterns generated by numerical iteration of the analytical model equations closely reproduced the experimental results. Our analysis demonstrates that slow deactivation of the delayed rectifier current determines the observed variations in excitability during diastole, whereas the inward rectifier current determines the amplitude and shape of the subthreshold response. Both currents combined are responsible for the development of Wenckebach periodicities in the ventricular cell. The overall study provides new insight into the ionic mechanisms of rate-dependent conduction block processes and may have important clinical implications as well.

The cellular mechanisms of Wenckebach periodicity were investigated in single, enzymatically dissociated guinea pig ventricular myocytes, as well as in computer reconstructions of transmembrane potential of the ventricular cell. When depolarizing current pulses of the appropriate magnitude were delivered repetitively to a well-polarized myocyte, rate-dependent activation failure was observed. Such behavior accurately mimicked the Wenckebach phenomenon in cardiac activation and was the consequence of variations in cell excitability during the diastolic phase of the cardiac cycle. The recovery of cell excitability during diastole was studied through the application of single test pulses of fixed amplitude and duration at variable delays with respect to a basic train of normal action potentials. The results show that recovery of excitability is a slow process that can greatly outlast action potential duration (i.e., postrepolarization refractoriness). Two distinct types of subthreshold responses were recorded when activation failure occurred: one was tetrodotoxin- and cobalt-insensitive (type 1) and the other was sensitive to sodium-channel blockade (type 2). Type 1 responses, which were commonly associated with the typical structure of the Wenckebach phenomenon (Mobitz type 1 block), were found to be the result of the nonlinear conductance properties of the inward rectifier current, IK1. Type 2 sodium-channel-mediated responses were associated with the so-called "millisecond Wenckebach." These responses may be implicated in the mechanism of Mobitz type 2 rate-dependent block. Single-cell voltage-clamp experiments suggest that variations in excitability during diastole are a consequence of the slow deactivation kinetics of the delayed rectifier, IK. Computer simulations of the ventricular cell response to depolarizing current pulses reproduced very closely all the response patterns obtained in the experimental preparation. It is concluded that postrepolarization refractoriness and Wenckebach periodicity are properties of normal cardiac excitable cells and can be explained in terms of the voltage dependence and slow kinetics of potassium outward currents. The conditions for the occurrence of intermittent activation failure during diastole will depend on the frequency and magnitude of the driving stimulus.

The effects of dantrolene sodium on dog Purkinje fibers, cat atrial and ventricular muscles were studied. Action potential duration was significantly increased and contractility was significantly decreased by dantrolene in all three types of tissue. The plateau phase of Purkinje fiber and occasionally atrial action potential was slightly depressed. Dantrolene sodium had no significant effect on resting membrane potential, action potential amplitude or upstroke velocity of phase 6. The negative inotropic effects were most pronounced in Purkinje fibers, followed by atrial muscle while papillary muscles were least sensitive. Contractile force of Purkinje fibers was decreased by relatively the same amount at all frequencies of stimulation. At faster rates, atrial and ventricular muscle contractility was depressed relatively more than at slower rates. Slow response action potentials in cat papillary muscle were diminished slightly, but this effect was not significant. All drug effects took 10 to 15 min to develop, reached a steady state after 30 to 40 min, and were irreversible upon washout. Increasing the extracellular calcium concentration reversed the dantrolene-induced changes. These findings suggest that effects of dantrolene are mediated in part by a decrease in the intracellular free calcium concentration in cardiac tissue.