Computer three-dimensional reconstruction of the atrioventricular node.

Because of its complexity, the atrioventricular node (AVN), remains 1 of the least understood regions of the heart. The aim of the study was to construct a detailed anatomic model of the AVN and relate it to AVN function. The electric activity of a rabbit AVN preparation was imaged using voltage-dependent dye. The preparation was then fixed and sectioned. Sixty-five sections at 60- to 340-microm intervals were stained for histology and immunolabeled for neurofilament (marker of nodal tissue) and connexin43 (gap junction protein). This revealed multiple structures within and around the AVN, including transitional tissue, inferior nodal extension, penetrating bundle, His bundle, atrial and ventricular muscle, central fibrous body, tendon of Todaro, and valves. A 3D anatomically detailed mathematical model (approximately 13 million element array) of the AVN and surrounding atrium and ventricle, incorporating all cell types, was constructed. Comparison of the model with electric activity recorded in experiments suggests that the inferior nodal extension forms the slow pathway, whereas the transitional tissue forms the fast pathway into the AVN. In addition, it suggests the pacemaker activity of the atrioventricular junction originates in the inferior nodal extension. Computer simulation of the propagation of the action potential through the anatomic model shows how, because of the complex structure of the AVN, reentry (slow-fast and fast-slow) can occur. In summary, a mathematical model of the anatomy of the AVN has been generated that allows AVN conduction to be explored.

Effect of electroporation on cardiac electrophysiology.

Defibrillation shocks are commonly used to terminate life-threatening arrhythmias. According to the excitation theory of defibrillation, such shocks are aimed at depolarizing the membranes of most cardiac cells, resulting in resynchronization of electrical activity in the heart. If shock-induced transmembrane potentials are large enough, they can cause transient tissue damage due to electroporation. In this review, evidence is presented that electroporation of the heart tissue can occur during clinically relevant intensities of the external electrical field and that electroporation can affect the outcome of defibrillation therapy, being both pro- and antiarrhythmic.Here, we present experimental evidence for electroporation in cardiac tissue, which occurs above a threshold of 25 V/cm as evident from propidium iodide uptake, transient diastolic depolarization, and reductions of action potential amplitude and its derivative. These electrophysiological changes can induce tachyarrhythmia, due to conduction block and possibly triggered activity; however, our findings provide the foundation for future design of effective methods to deliver genes and drugs to cardiac tissues, while avoiding possible side effects such as arrhythmia and mechanical stunning.

Autonomic control and innervation of the atrioventricular junctional pacemaker.

BACKGROUND: The main physiologic function of the AV junction is control of timing between atrial and ventricular excitation. However, under pathologic conditions, the AV junction may become the pacemaker of the heart. Unlike the well-characterized sinoatrial node (SAN), autonomic control of the AV junctional pacemaker has not been studied. OBJECTIVE: The purpose of this study was to characterize the autonomic control and innervation of the AV junctional pacemaker. METHODS: The response of rabbit AV junctional pacemaker to autonomic stimulation was investigated using optical mapping, autonomic modulation via subthreshold stimulation (n = 12), and quantitative immunohistochemistry (n = 5), and the density of parasympathetic and sympathetic innervation in optically mapped preparations was quantified. RESULTS: Subthreshold stimulation applied adjacent to the conduction system in the triangle of Koch autonomically modulates the junctional rate, and parasympathetic and sympathetic components can be separated with atropine and the beta-blocker nadolol. Subthreshold stimulation increased the rate maximally to 2.1 +/- 0.4 times when applied with atropine. Unlike the SAN pacemaker, which shifts significantly in response to autonomic stimulation, the AV junctional pacemaker remains stationary (most often in the inferior nodal extension), moving in only 5% of subthreshold stimulation trials. Staining with tyrosine hydroxylase and choline acetyltransferase revealed heterogeneous innervation within the AV junction. CONCLUSION: AV junctional rhythm can be autonomically modulated with subthreshold stimulation to produce junctional rates of 145 +/- 16 bpm (cycle length 412 +/- 29 ms), similar to sinus rates in rabbit. Unlike the SAN, the anatomic location of the AV junctional pacemaker is stable during autonomic modulation.

Three-dimensional panoramic imaging of cardiac arrhythmias in rabbit heart.

Cardiac fluorescent optical imaging provides the unique opportunity to investigate the dynamics of propagating electrical waves during ventricular arrhythmias and the termination of arrhythmias by strong electric shocks. Panoramic imaging systems using charge-coupled device (CCD) cameras as the photodetector have been developed to overcome the inability to monitor electrical activity from the entire cardiac surface. Photodiode arrays (PDAs) are known to have higher temporal resolution and signal quality, but lower spatial resolution compared to CCD cameras. We construct a panoramic imaging system with three PDAs and image Langendorff perfused rabbit hearts (n=18) during normal sinus rhythm, epicardial pacing, and arrhythmias. The recorded spatiotemporal dynamics of electrical activity is texture mapped onto a reconstructed 3-D geometrical heart model specific to each heart studied. The PDA-based system provides sufficient spatial resolution (1.72 mm without interpolation) for the study of wavefront propagation in the rabbit heart. The reconstructed 3-D electrical activity provides us with a powerful tool to investigate the fundamental mechanisms of arrhythmia maintenance and termination.

Atrioventricular conduction with and without AV nodal delay: two pathways to the bundle of His in the rabbit heart.

The electrophysiological properties of atrioventricular (AV) nodal dual pathways have traditionally been investigated with premature stimuli delivered with right atrial pacing. However, little is known about the functional characteristics of AV nodal inputs outside of this context. Superfused rabbit triangle of Koch preparations (n = 8) and Langendorff-perfused hearts (n = 10) were paced throughout the triangle of Koch and mapped electrically and optically for activation pattern, electrogram and optical action potential morphologies, stimulation thresholds, and stimulus-His (S-H) intervals. Optical mapping and changes in His electrogram morphology were used to confirm the activation pathway. Pacing stimuli >or=2 mm above the tricuspid valve caused fast-pathway activation of the AV node and His with a threshold of 2.4 +/- 1.6 mA. An area directly below the coronary sinus had high thresholds (8.6 +/- 1.4 mA) that also resulted in fast-pathway excitation (P < 0.001). S-H intervals (81 +/- 19 ms) for fast-pathway activation remained constant throughout the triangle of Koch, reflecting the AV delay. Stimuli applied <2 mm from the tricuspid valve resulted in slow pathway (SP) excitation or direct His excitation (4.4 +/- 2.2 mA threshold; P < 0.001 compared with fast pathway). For SP/His pacing, S-H intervals showed a strong dependence on the distance from the His electrode and were significantly lower than S-H intervals for fast-pathway activation. SP/His pacing also displayed characteristic changes in His electrogram morphology. In conclusion, optical maps and S-H intervals for SP/His activation suggest that AV conduction via SP bypasses the compact AV node via the lower nodal bundle, which may be utilized to achieve long-term ventricular synchronization.

Direct measurements of membrane time constant during defibrillation strength shocks.

BACKGROUND: Defibrillation shocks impose significant energy demand on implantable cardioverter-defibrillators (ICDs). Several modeling studies have been devoted to optimizing shock parameters, and a large number of these studies treat the heart as a simplified lumped network. The time constant of membrane polarization (tau(m)) is a key variable for such modeling efforts. OBJECTIVE: The purpose of this study was to perform direct measurements of transmembrane potential (V(m)) during defibrillation strength shocks and estimate tau(m) of membrane polarization. METHODS: A portion of the left ventricular epicardial surface of Langendorff-perfused rabbit hearts was stimulated using uniform electric fields produced by two parallel line electrodes. The V(m)s were recorded from di-4-ANEPPS-stained hearts using a multisite optical mapping system. The hearts were paced with 20 S1 pulses from the apex, and shocks (S2: 5, 10, 20 V/cm) were applied via the line electrodes during the action potential of the 20th S1 at two different coupling intervals (S1S2: 120 and 180 ms). Residual responses were obtained by subtracting responses to the 19th S1 from the responses to the 20th S1S2 pair and used for time-constant analysis by fitting a monoexponential function. RESULTS: tau(m) exhibited a large variation and ranged from approximately 1 to 30 ms. Furthermore, tau(m) varied with electric field strength, S1S2 interval, position of the tissue from stimulating electrodes, and polarity of the response. To a large extent, the effects of all these factors were captured in a single parameter-the change in transmembrane voltage (DeltaV(m)) in response to the applied field (E). tau(m) showed a monotonically decreasing trend with DeltaV(m) for all Es and S1S2s. CONCLUSION: Time constant of membrane polarization varies significantly during defibrillation strength shocks and shows a strong dependence on DeltaV(m).

Gene printer: laser-scanning targeted transfection of cultured cardiac neonatal rat cells.

Heterogeneous gene expression in cardiac cells and tissues which requires targeted delivery of foreign DNA into selected cells or regions is needed for the development of novel therapies. Several techniques have been employed for targeted transfection, such as direct microinjection into cells or targeted electroporation. However, these techniques have limited bandwidth or spatial resolution of transfection. We aimed to develop a method for transfection of cardiac cells by means of laser-assisted optoporation using a standard confocal microscope. This technique allows for the transfection of selected cell types in the presence of other cell types as long as they are distinguishable with a microscope. This technique can work as a "gene printer" creating arbitrarily shaped areas of transfected cells.

Mechanisms of unpinning and termination of ventricular tachycardia.

High-energy defibrillation shock is the only therapy for ventricular tachyarrhythmias. However, because of adverse side effects, lowering defibrillation energy is desirable. We investigated mechanisms of unpinning, destabilization, and termination of ventricular tachycardia (VT) by low-energy shocks in isolated rabbit right ventricular preparations (n = 22). Stable VT was initiated with burst pacing and was optically mapped. Monophasic "unpinning" shocks (10 ms) of different strengths were applied at various phases throughout the reentry cycle. In 8 of 22 preparations, antitachycardia pacing (ATP: 8-20 pulses, 50-105% of period, 0.8-10 mA) was also applied. Termination of reentry by ATP was achieved in only 5 of 8 preparations. Termination by unpinning occurred in all 22 preparations. Rayleigh's test showed a statistically significant unpinning phase window, during which reentry could be unpinned and subsequently terminated with E80 (magnitude at which 80% of reentries were unpinned) = 1.2 V/cm. All reentries were unpinned with field strengths < or = 2.4 V/cm. Unpinning was achieved by inducing virtual electrode polarization and secondary sources of excitation at the core of reentry. Optical mapping revealed the mechanisms of phase-dependent unpinning of reentry. These results suggest that a 20-fold reduction in energy could be achieved compared with conventional high-energy defibrillation and that the unpinning method may be more effective than ATP for terminating stable, pinned reentry in this experimental model.

Optical mapping of the atrioventricular junction.

In the normal heart, the atrioventricular node (AVN) is part of the sole pathway between the atria and ventricles, and is responsible for the appropriate atrial-ventricular delay. Under normal physiological conditions, the AVN controls appropriate frequency-dependent delay of contractions. The AVN also plays an important role in pathology: it protects ventricles during atrial tachyarrhythmia, and during sinoatrial node failure the atrioventricular (AV) junction assumes the role of pacemaker. Finally, the AV junction provides an anatomic substrate for AV nodal reentrant tachycardia, which is the most prevalent supraventricular tachycardia in humans. Using fluorescent imaging with voltage-sensitive dye and immunohistochemistry, we have investigated the structure-function relationship of the atrioventricular (AV) junction during normal conduction, reentry, and junctional rhythm. We identified the site of origin of junctional rhythm at the posterior extension of the AV node (AVN) in 78% (n=23) of the studied hearts and we found that this pacemaker is sensitive to autonomic control. For instance, when the autonomic nervous system was activated using subthreshold stimulation, a transient accelerated junctional rhythm was observed when subthreshold stimulation was terminated. A very similar phenomenon is observed clinically during slow pathway ablations treating AV nodal reentrant tachycardia (AVNRT). The autonomic control of the AV junction was investigated using immunohistochemistry, showing that the AV junction of the rabbit is very densely innervated with both cholinergic and adrenergic neurons. The posterior AV nodal extension was similar to the compact AVN as determined by morphologic and molecular investigations. In particular, both the posterior extension and the compact node express the pacemaking channel HCN4 (responsible for the IF current) and neurofilament 160. In the rabbit heart, AV junction conduction, reentrant arrhythmia, and spontaneous rhythm are governed by heterogeneity of expression of several isoforms of gap junctions and ion channels, and these properties are regulated by the autonomic nervous system. Uniform neurofilament expression suggests that AV nodal posterior extensions are an integral part of the cardiac pacemaking and conduction system.

Fluorescence imaging for real-time monitoring of high-intensity focused ultrasound cardiac ablation.

Side effects and limitations of radio-frequency ablation of cardiac arrhythmias prompted search for alternative energy sources and means of their application. High-intensity focused ultrasound (HIFU) is becoming an increasingly attractive modality for ablation because of its unique ability for non-invasive or minimally invasive, non-contact focal ablation in 3D volume without affecting intervening and surrounding cells. The purpose of this study is to develop a real-time monitoring technique to elucidate HIFU-induced modifications of electrical conduction in cardiac tissues and to investigate the HIFU cardiac ablation process to help to achieve optimal HIFU ablation outcome. We conducted experimental studies applying HIFU at 4.23 MHz to ablate the atrio-ventricular (AV) node and ventricular tissue of Langendorff-perfused rabbit hearts. We employed fluorescent voltage-sensitive dye imaging and surface electrodes to monitor the electrical conduction activity induced by HIFU application in real time. In ventricular epicardium HIFU ablation, fluorescent imaging revealed gradual reduction of the plateau phase and amplitude of the action potential. Subsequently, conduction block and cell death were observed at the site of ablation. When HIFU was applied to the AV node, fluorescent imaging and electrograms revealed the development of the AV block. The study establishes that real-time fluorescent imaging provides novel monitoring and assessment to study HIFU cardiac ablation, which may be able to provide improved understanding of HIFU cardiac ablation process and mechanism useful for development of successful clinical applications.

Hibernator Citellus undulatus maintains safe cardiac conduction and is protected against tachyarrhythmias during extreme hypothermia: possible role of Cx43 and Cx45 up-regulation.

BACKGROUND: Most mammals experience cardiac arrest during hypothermia. In contrast, hibernators remain in sinus rhythm even at body temperatures of 0 degrees C. OBJECTIVES: The purpose of this study was to quantify electrical activity and connexin expression in the heart of hibernating Siberian ground squirrel Citellus undulatus. METHODS: Optical imaging and microelectrode recordings were conducted in Langendorff-perfused hearts and isolated papillary muscles of summer active (SA, n = 19), winter hibernating (WH, n = 21), interbout arousal (IBA, n = 12), and winter active (WA, n = 3) ground squirrels and rabbits (n = 14) at temperatures from +37 degrees C to +3 degrees C. RESULTS: All studied SA and WH hearts maintained spontaneous sinus rhythm, safe propagation through the entire conduction system, and normal pattern of ventricular excitation at all temperatures. However, three of the seven IBA and all rabbit hearts lost excitability at 10 degrees C +/- 1 degrees C and 12 degrees C +/- 1 degrees C, respectively. In WH, SA, and IBA ground squirrels, temperature reduction from 37 degrees C to 3 degrees C resulted in a 10-fold slowing of ventricular conduction velocity and increased excitation threshold. At any temperature, WH ventricles had faster conduction velocity and lower excitation threshold compared with SA and IBA. Immunolabeling demonstrated that connexin43 (Cx43) was significantly up-regulated in WH and WA compared with SA myocardium: Cx43 area density was 12.4 +/- 1.3, 15.0 +/- 3.0 and 8.6 +/- 1.1 microm(2)/1,000 microm(2), respectively. Moreover, Cx45 was expressed in the WH but not in the SA or WA ventricles. CONCLUSION: Hibernator Citellus undulatus has evolved to maintain safe conduction at extreme hypothermia via up-regulation of Cx43 and Cx45 in order to protect the heart against arrhythmia associated with hypothermia.

Electroporation of the heart.

Defibrillation shocks are commonly used to terminate life-threatening arrhythmias. According to the excitation theory of defibrillation, such shocks are aimed at depolarizing the membranes of most cardiac cells resulting in resynchronization of electrical activity in the heart. If shock-induced changes in transmembrane potential are large enough, they can cause transient tissue damage due to electroporation. In this review evidence is presented that (a) electroporation of the heart tissue can occur during clinically relevant intensities of the external electrical field, and (b) electroporation can affect the outcome of defibrillation therapy; being both pro- and anti-arrhythmic.

The Gurvich waveform has lower defibrillation threshold than the rectilinear waveform and the truncated exponential waveform in the rabbit heart.

Implantable cardioverter defibrillator studies have established the superiority of biphasic waveforms over monophasic waveforms. However, external defibrillator studies of biphasic waveforms are not as widespread. Our objective was to compare the defibrillation efficacy of clinically used biphasic waveforms, i.e., truncated exponential, rectilinear, and quasi-sinusoidal (Gurvich) waveforms in a fibrillating heart model. Langendorff-perfused rabbit hearts (n = 10) were stained with a voltage-sensitive fluorescent dye, Di-4-ANEPPS. Transmembrane action potentials were optically mapped from the anterior epicardium. We found that the Gurvich waveform was significantly superior (p < 0.05) to the rectilinear and truncated exponential waveforms. The defibrillation thresholds (mean +/- SE) were as follows: Gurvich, 0.25 +/- 0.01 J; rectilinear-1, 0.34 +/- 0.01 J; rectilinear-2, 0.33 +/- 0.01 J; and truncated exponential, 0.32 +/- 0.02 J. Using optically recorded transmembrane responses, we determined the shock-response transfer function, which allowed us to predict the cellular response to waveforms at high accuracy. The passive parallel resistor-capacitor model (RC-model) predicted polarization superiority of the Gurvich waveform in the myocardium with a membrane time constant (taum) of less than 2 ms. The finding of a lower defibrillation threshold with the Gurvich waveform in an in vitro model of external defibrillation suggests that the Gurvich waveform may be important for future external defibrillator designs.

Mechanisms of superiority of ascending ramp waveforms: new insights into mechanisms of shock-induced vulnerability and defibrillation.

Monophasic ascending ramp (AR) and descending ramp (DR) waveforms are known to have significantly different defibrillation thresholds. We hypothesized that this difference arises due to differences in mechanisms of arrhythmia induction for the two waveforms. Rabbit hearts (n = 10) were Langendorff perfused, and AR and DR waveforms (7, 20, and 40 ms) were randomly delivered from two line electrodes placed 10 mm apart on the anterior ventricular epicardium. We optically mapped cellular responses to shocks of various strengths (5, 10, and 20 V/cm) and coupling intervals (CIs; 120, 180, and 300 ms). Optical mapping revealed that maximum virtual electrode polarization (VEP) was reached at significantly different times for AR and DR of the same duration (P < 0.05) for all tested CIs. As a result, VEP for AR were stronger than for DR at the end of the shock. Postshock break excitation resulting from AR generated faster propagation and typically could not form reentry. In contrast, partially dissipated VEP resulting from DR generated slower propagation; the wavefront was able to propagate into deexcited tissue and thus formed a shock-induced reentry circuit. Therefore, for the same delivered energy, AR was less proarrhythmic compared with DR. An active bidomain model was used to confirm the electrophysiological results. The VEP hypothesis explains differences in vulnerability associated with monophasic AR and DR waveforms and, by extension, the superior defibrillation efficacy of the AR waveform compared with the DR waveform.

Structure-function relationship in the AV junction.

In the normal heart, the atrioventricular node (AVN) is part of the sole pathway between the atria and ventricles. Under normal physiological conditions, the AVN controls appropriate frequency-dependent delay of contractions. The AVN also plays an important role in pathology: it protects ventricles during atrial tachyarrhythmia, and during sinoatrial node failure an AV junctional pacemaker can drive the heart. Finally, the AV junction provides an anatomical substrate for reentry. Using fluorescent imaging with voltage-sensitive dyes and immunohistochemistry, we have investigated the structure-function relationship of the AV junction during normal conduction, reentry, and junctional rhythm. We identified molecular and structural heterogeneity that provides a substrate for the dual-pathway AVN conduction. We observed heterogeneity of expression of three isoforms of connexins: Cx43, Cx45, and Cx40. We identified the site of origin of junctional rhythm at the posterior extension of the AV node in 79% (n = 14) of the studied hearts. This structure was similar to the compact AV node as determined by morphologic and molecular investigations. In particular, both the posterior extension and the compact node express the pacemaking channel HCN4 (responsible for the I(F) current) and neurofilament 160. In the rabbit heart, AV junction conduction, reentrant arrhythmia, and spontaneous rhythm are governed by heterogeneity of expression of several isoforms of gap junctions and ion channels. Uniform neurofilament expression suggests that AV nodal posterior extensions are an integral part of the cardiac pacemaking and conduction system. On the other hand, differential expression of Cx isoforms in this region provides an explanation of longitudinal dissociation, dual-pathway electrophysiology, and AV nodal reentrant arrhythmogenesis.

Optical imaging of the heart.

Optical techniques have revolutionized the investigation of cardiac cellular physiology and advanced our understanding of basic mechanisms of electrical activity, calcium homeostasis, and metabolism. Although optical methods are widely accepted and have been at the forefront of scientific discoveries, they have been primarily applied at cellular and subcellular levels and considerably less to whole heart organ physiology. Numerous technical difficulties had to be overcome to dynamically map physiological processes in intact hearts by optical methods. Problems of contraction artifacts, cellular heterogeneities, spatial and temporal resolution, limitations of surface images, depth-of-field, and need for large fields of view (ranging from 2x2 mm2 to 3x3 cm2) have all led to the development of new devices and optical probes to monitor physiological parameters in intact hearts. This review aims to provide a critical overview of current approaches, their contributions to the field of cardiac electrophysiology, and future directions of various optical imaging modalities as applied to cardiac physiology at organ and tissue levels.

Virtual electrode theory explains pacing threshold increase caused by cardiac tissue damage.

The virtual electrode polarization (VEP) effect is believed to play a key role in electrical stimulation of heart muscle. However, under certain conditions, including clinically, its existence and importance remain unknown. We investigated the influence of acute tissue damage produced by continuous pacing with strong current (40-mA, 4-ms biphasic pulses with 4-Hz frequency for 5 min) on stimulus-generated VEPs and pacing thresholds. A fluorescent optical mapping technique was used to obtain stimulus-induced transmembrane potential distribution around a pacing electrode applied to the ventricular surface of a Langendorff-perfused rabbit heart (n = 5). Maps and pacing thresholds were recorded before and after tissue damage. Spatial extents of electroporation and cell uncoupling were assessed by propidium iodide (n = 2) and connexin43 (n = 3) antibody staining, respectively. On the basis of these data, passive and active three-dimensional bidomain models were built to determine VEP patterns and thresholds for different-sized areas of the damaged region. Electrophysiological results showed that acute tissue damage led to disappearance of the VEP with an associated significant increase in pacing thresholds. Damage was expressed in electroporation and cell uncoupling within an approximately 1.0-mm-diameter area around the tip of the electrode. According to computer simulations, cell uncoupling, rather than electroporation, might be the direct cause of VEP elimination and threshold increase, which was nonlinearly dependent on the size of the damaged region. Fiber rotation with depth did not substantially affect the numerical results. The study explains failure to stimulate damaged tissue within the concepts of the VEP theory.

Site of origin and molecular substrate of atrioventricular junctional rhythm in the rabbit heart.

During failure of the sinoatrial node, the heart can be driven by an atrioventricular (AV) junctional pacemaker. The position of the leading pacemaker site during AV junctional rhythm is debated. In this study, we present evidence from high-resolution fluorescent imaging of electrical activity in rabbit isolated atrioventricular node (AVN) preparations that, in the majority of cases (11 out of 14), the AV junctional rhythm originates in the region extending from the AVN toward the coronary sinus along the tricuspid valve (posterior nodal extension, PNE). Histological and immunohistochemical investigation showed that the PNE has the same morphology and unique pattern of expression of neurofilament160 (NF160) and connexins (Cx40, Cx43, and Cx45) as the AVN itself. Block of the pacemaker current, If, by 2 mmol/L Cs+ increased the AV junctional rhythm cycle length from 611+/-84 to 949+/-120 ms (mean+/-SD, n=6, P<0.001). Immunohistochemical investigation showed that the principal If channel protein, HCN4, is abundant in the PNE. As well as the AV junctional rhythm, the PNE described in this study may also be involved in the slow pathway of conduction into the AVN as well as AVN reentry, and the predominant lack of expression of Cx43 as well as the presence of Cx45 in the PNE shown could help explain its slow conduction.

Nonlinear effects in subthreshold virtual electrode polarization.

Introduction of the virtual electrode polarization (VEP) theory suggested solutions to several century-old puzzles of heart electrophysiology including explanation of the mechanisms of stimulation and defibrillation. Bidomain theory predicts that VEPs should exist at any stimulus strength. Although the presence of VEPs for strong suprathreshold pulses has been well documented, their existence at subthreshold strengths during diastole remains controversial. We studied cardiac membrane polarization produced by subthreshold stimuli in 1) rabbit ventricular muscle using high-resolution fluorescent imaging with the voltage-sensitive dye pyridinium 4-[2-[6-(dibutylamino)-2-naphthalenyl]-ethenyl]-1-(3-sulfopropyl)hydroxide (di-4-ANEPPS) and 2) an active bidomain model with Luo-Rudy ion channel kinetics. Both in vitro and in numero models show that the common dog-bone-shaped VEP is present at any stimulus strength during both systole and diastole. Diastolic subthreshold VEPs exhibited nonlinear properties that were expressed in time-dependent asymmetric reversal of membrane polarization with respect to stimulus polarity. The bidomain model reveals that this asymmetry is due to nonlinear properties of the inward rectifier potassium current. Our results suggest that active ion channel kinetics modulate the transmembrane polarization pattern that is predicted by the linear bidomain model of cardiac syncytium.