Biomedical subjects

Leslie Tung

Publications and source records attributed to Leslie Tung.

At least 19 recordsLinked to original sources

PubMed2006

Spiral wave attachment to millimeter-sized obstacles.

BACKGROUND: Functional reentry in the heart takes the form of spiral waves. Drifting spiral waves can become pinned to anatomic obstacles and thus attain stability and persistence. Lidocaine is an antiarrhythmic agent commonly used to treat ventricular tachycardia clinically. We examined the ability of small obstacles to anchor spiral waves and the effect of lidocaine on their attachment. METHODS AND RESULTS: Spiral waves were electrically induced in confluent monolayers of cultured, neonatal rat cardiomyocytes. Small, circular anatomic obstacles (0.6 to 2.6 mm in diameter) were situated in the center of the monolayers to provide an anchoring site. Eighty reentry episodes consisting of at least 4 revolutions were studied. In 36 episodes, the spiral wave attached to the obstacle and became stationary and sustained, with a shorter reentry cycle length and higher rate. Spiral waves could attach to obstacles as small as 0.6 mm, with a likelihood for attachment that increased with obstacle size. After attachment, both conduction velocity of the wave-front tip and wavelength near the obstacle adapted from their pre-reentry values and increased linearly with obstacle size. In contrast, reentry cycle length did not correlate significantly with obstacle size. Addition of lidocaine 90 mumol/L depressed conduction velocity, increased reentry cycle length, and caused attached spiral waves to become quasi- attached to the obstacle or terminate. CONCLUSIONS: Anchored spiral waves exhibit properties of both unattached spiral waves and anatomic reentry. Their behavior may be representative of functional reentry dynamics in cardiac tissue, particularly in the setting of monomorphic tachyarrhythmias.

Animals↗

PubMed2006

Electrical pacing counteracts intrinsic shortening of action potential duration of neonatal rat ventricular cells in culture.

Previous studies have demonstrated the relationship between the functional electrophysiological properties of cultured neonatal rat ventricular myocytes (NRVMs) and the ability of the substrate to induce and sustain arrhythmia. The goal of this study was to examine the effects of chronic pacing at a constant rate akin to that in vivo, on the functional electrophysiological properties of NRVM monolayers. Confluent NRVM monolayers grown on 20 mm diameter cover slips were left either unpaced or were stimulated at 3 Hz for the duration of the culture, and were optically mapped on days 4, 6, or 8. Action potential duration at 80% repolarization (APD80), conduction velocity (CV), and Kv4.3 (Ito) and NCX protein expression were measured. The effects of the excitation-contraction uncoupler 2,3-butadione monoxime (BDM) were also investigated. The 2 Hz APD80 of non-paced monolayers decreased significantly on days 6 (137.1+/-13.9 ms) and 8 (109.8+/-9.0 ms) compared with day 4 (197.0+/-11.8 ms), while that of paced monolayers did not (206.8+/-9.7, 209.1+/-9.2, and 210.6+/-9.9 ms, respectively). The 2 Hz CV of non-paced monolayers increased significantly on days 6 (26.0+/-1.6 cm/s) and 8 (26.5+/-1.0 cm/s) compared with day 4 (20.0+/-1.0 cm/s), while that of paced monolayers did not change significantly (26.0+/-2.0, 26.0+/-1.0, and 23.8+/-1.2 cm/s, respectively). The restitution curves of APD80 and CV of paced monolayers were also unchanging from days 4 through 8. Despite the unchanging APD80 and CV, a decrease in Kv4.3 expression and an increase in NCX expression were observed in paced compared with non-paced monolayers. Cessation of pacing or administration of BDM caused a reversal of phenotype back to that of non-paced monolayers. In summary, chronic electrical stimulation of confluent NRVM monolayers results in stabilization of APD80 and an advancement of the developmental rise of CV that is mediated by electromechanical coupling. These effects produce a steadier functional phenotype that may be beneficial for electrophysiological studies.

Action Potentials↗

PubMed2006

Proarrhythmic potential of mesenchymal stem cell transplantation revealed in an in vitro coculture model.

BACKGROUND: Mesenchymal stem cells (MSCs) are bone marrow stromal cells that are in phase 1 clinical studies of cellular cardiomyoplasty. However, the electrophysiological effects of MSC transplantation have not been studied. Although improvement of ventricular function would represent a positive outcome of MSC transplantation, focal application of stem cells has the potential downside of creating inhomogeneities that may predispose the heart to reentrant arrhythmias. In the present study we use an MSC and neonatal rat ventricular myocyte (NRVM) coculture system to investigate potential proarrhythmic consequences of MSC transplantation into the heart. METHODS AND RESULTS: Human MSCs were cocultured with NRVMs in ratios of 1:99, 1:9, and 1:4 and optically mapped. We found that conduction velocity was decreased in cocultures compared with controls, but action potential duration (APD80) was not affected. Reentrant arrhythmias were induced in 86% of cocultures containing 10% and 20% MSCs (n=36) but not in controls (n=7) or cocultures containing only 1% MSCs (n=4). Immunostaining, Western blot, and dye transfer revealed the presence of functional gap junctions involving MSCs. CONCLUSIONS: Our results suggest that mixtures of MSCs and NRVMs can produce an arrhythmogenic substrate. The mechanism of reentry is probably increased tissue heterogeneity resulting from electric coupling of inexcitable MSCs with myocytes.

Animals↗

PubMed2006

Structure-related initiation of reentry by rapid pacing in monolayers of cardiac cells.

This study examines how a zigzag pattern of conduction, a form of structural heterogeneity frequently found in old or diseased hearts, affects the vulnerability to reentry during rapid pacing. A central rectangular island (8x4 mm) containing a predefined zigzag pattern was created in cultured isotropic monolayers of neonatal rat ventricular myocytes. Impulse propagation was optically mapped from 253 sites using voltage-sensitive dye and was anisotropic within the zigzag island. With increasing interval between neighboring transverse connections (a), relative to the distance between longitudinal strands (b), transverse conduction velocity (CV) decreased to 66+/-6%, 20+/-2%, and 15+/-2% of CV in the surrounding isotropic region, whereas longitudinal CV increased to 102+/-8%, 113+/-12%, and 131+/-23% for a:b ratios of 1:1, 1:5, and 1:9, respectively. During rapid pacing, propagation distal to the island was steered from the side of the island with more transverse connections ("dominant" side) toward the side with fewer connections ("weak" side). Increased asymmetry in the pattern accentuated this effect, and resulted in increased rate-dependent differences in CV on the 2 sides. Consequently, a functional obstacle formed on the weak side, followed by development of single loop reentry. The reentrant wave revolved around a line of block defined by the border of the island. Reentry chirality was determined by the weak side location, and the pacing rate needed to initiate reentry decreased with increased asymmetry in the pattern. In conclusion, reentry is readily induced by rapid pacing in confluent cardiac cell monolayers containing a central and asymmetric island of zigzag conduction.

Action Potentials↗

PubMed2006

Optical imaging of arrhythmias in tissue culture.

BACKGROUND: Cardiac cell cultures are becoming an important experimental system of minimal complexity that captures many of the salient features of myocardial tissue function and are simple enough that tissue parameters can be controlled systematically. Fundamental mechanisms that underlie normal and pathological electrophysiology at the tissue level can be studied. Of particular interest are spiral waves, which underlie many tachyarrhythmias and fibrillation. METHODS: Methods of patterned growth were used to control tissue structure, and contact fluorescence imaging was used to visualize the spread of electrical waves in confluent monolayers of neonatal rat ventricular cells stained with voltage-sensitive dye. RESULTS: Work is summarized regarding anisotropy, multiarmed spirals, cocultures of cardiac cells and skeletal myoblasts or mesenchymal stem cells, mechanical excitation, attachment of spiral waves to small anatomical obstacles, perturbation of spiral waves by external electric fields, and structure-based facilitation of spiral wave formation. CONCLUSIONS: The cultured cell monolayer is a contemporary experimental model encompassing great versatility for basic studies of wavefront propagation and cardiac arrhythmias.

Action Potentials↗

PubMed2005

Acceleration of functional reentry by rapid pacing in anisotropic cardiac monolayers: formation of multi-wave functional reentries.

OBJECTIVE: Attempts to cardiovert tachycardia by rapid point pacing can sometimes result in transient or stable increase of the heart rate (acceleration), changed ECG morphology, and/or fibrillation. The goal of this study was to investigate the effect of rapid pacing on the dynamics of functional reentry in monolayer cultures of cardiac cells. METHODS: Fully confluent, uniformly anisotropic monolayers of neonatal rat ventricular myocytes were prepared using methods of microabrasion. Cells were paced by a point electrode at rest and during functional reentry, and membrane voltages were optically mapped. RESULTS: Point pacing readily induced single loop anisotropic functional reentry with monomorphic optical pseudo-ECG (pECG) and average rotation period of 193+/-52 ms (n=71 monolayers). Attempts to cardiovert reentry by rapid pacing at rates 10-50% faster than the reentry rate were successful in 57/71 monolayers. In 14/71 monolayers, the number of rotating waves was stably increased by 1 to 4, yielding a 10-70% acceleration of pECG rate and change to a different monomorphic or polymorphic pECG. The resulting multi-wave functional reentries were classified based on the number and direction of their rotating waves. The higher the number of waves in the multi-wave reentry, the more accelerated was the rate of cell firing in the monolayer. Importantly, stable acceleration was only inducible in monolayers with relatively deep and broad conduction velocity restitution relationships. Reapplication of point pacing further accelerated, decelerated, or eventually terminated the reentrant activity. CONCLUSIONS: These results suggest that stable multiplication of rotating waves in conjunction with a deep and broad conduction velocity restitution relationship is a possible mechanism for stable acceleration of functional reentry by rapid pacing.

Animals↗

PubMed2005

Antiarrhythmic engineering of skeletal myoblasts for cardiac transplantation.

Skeletal myoblasts are an attractive cell type for transplantation because they are autologous and resistant to ischemia. However, clinical trials of myoblast transplantation in heart failure have been plagued by ventricular tachyarrhythmias and sudden cardiac death. The pathogenesis of these arrhythmias is poorly understood, but may be related to the fact that skeletal muscle cells, unlike heart cells, are electrically isolated by the absence of gap junctions. Using a novel in vitro model of myoblast transplantation in cardiomyocyte monolayers, we investigated the mechanisms of transplant-associated arrhythmias. Cocultures of human skeletal myoblasts and rat cardiomyocytes resulted in reentrant arrhythmias (spiral waves) that reproduce the features of ventricular tachycardia seen in patients receiving myoblast transplants. These arrhythmias could be terminated by nitrendipine, an l-type calcium channel blocker, but not by the Na channel blocker lidocaine. Genetic modification of myoblasts to express the gap junction protein connexin43 decreased arrhythmogenicity in cocultures, suggesting a specific means for increasing the safety (and perhaps the efficacy) of myoblast transplantation in patients.

Action Potentials↗

PubMed2005

Mechanoelectrical excitation by fluid jets in monolayers of cultured cardiac myocytes.

Although the prevailing view of mechanoelectric feedback (MEF) in the heart is in terms of longitudinal cell stretch, other mechanical forces are considerable during the cardiac cycle, including intramyocardial pressure and shear stress. Their contribution to MEF is largely unknown. In this study, mechanical stimuli in the form of localized fluid jet pulses were applied to neonatal rat ventricular cells cultured as confluent monolayers. Such pulses result in pressure and shear stresses (but not longitudinal stretch) in the monolayer at the point of impingement. The goal was to determine whether these mechanical stimuli can trigger excitation, initiate a propagated wave, and induce reentry. Cells were stained with the voltage-sensitive dye RH237, and multi-site optical mapping was used to record the spread of electrical activity in isotropic and anisotropic monolayers. Pulses (10 ms) with velocities ranging from 0.3 to 1.8 m/s were applied from a 0.4-mm diameter nozzle located 1 mm above the cell monolayer. Fluid jet pulses resulted in circular wavefronts that propagated radially from the stimulus site. The likelihood for mechanical stimulation was quantified as an average stimulus success rate (ASSR). ASSR increased with jet amplitude and time waited between stimuli and decreased with the application of gadolinium and streptomycin, blockers of stretch-activated channels, but not with nifedipine, a blocker of the L-type Ca channel. Absence of cellular injury was confirmed by smooth propagation maps and propidium iodide stains. In rare instances, the mechanical pulse resulted in the induction of reentrant activity. We conclude that mechanical stimuli other than stretch can evoke action potentials, propagated activity, and reentrant arrhythmia in two-dimensional sheets of cardiac cells.

Animals↗

PubMed2005

Paradoxical loss of excitation with high intensity pulses during electric field stimulation of single cardiac cells.

Transmembrane potential responses of single cardiac cells stimulated at rest were studied with uniform rectangular field pulses having durations of 0.5-10 ms. Cells were enzymatically isolated from guinea pig ventricles, stained with voltage sensitive dye di-8-ANEPPS, and stimulated along their long axes. Fluorescence signals were recorded with spatial resolution of 17 microm for up to 11 sites along the cell. With 5 and 10 ms pulses, all cells (n = 10) fired an action potential over a broad range of field amplitudes (approximately 3-65 V/cm). With 0.5 and 1 ms pulses, all cells (n = 7) fired an action potential for field amplitudes ranging from the threshold value (approximately 4-8 V/cm) to 50-60 V/cm. However, when the field amplitude was further increased, five of seven cells failed to fire an action potential. We postulated that this paradoxical loss of excitation for higher amplitude field pulses is the result of nonuniform polarization of the cell membrane under conditions of electric field stimulation, and a counterbalancing interplay between sodium current and inwardly rectifying potassium current with increasing field strength. This hypothesis was verified using computer simulations of a field-stimulated guinea pig ventricular cell. In conclusion, we show that for stimulation with short-duration pulses, cells can be excited for fields ranging between a low amplitude excitation threshold and a high amplitude threshold above which the excitation is suppressed. These results can have implications for the mechanistic understanding of defibrillation outcome, especially in the setting of diseased myocardium.

Action Potentials↗

PubMed2004

Multiarm spirals in a two-dimensional cardiac substrate.

A variety of chemical and biological nonlinear excitable media, including heart tissue, can support stable, self-organized waves of activity in a form of rotating single-arm spirals. In particular, heart tissue can support stationary and meandering spirals of electrical excitation, which have been shown to underlie different forms of cardiac arrhythmias. In contrast to single-arm spirals, stable multiarm spirals (multiple spiral waves that rotate in the same direction around a common organizing center) have not been demonstrated and studied yet in living excitable tissues. Here, we show that persistent multiarm spirals of electrical activity can be induced in monolayer cultures of neonatal rat heart cells by a short, rapid train of electrical point stimuli applied during single-arm-spiral activity. Stable formation is accomplished only in monolayers that show a relatively broad and steep dependence of impulse wavelength and propagation velocity on rate of excitation. The resulting multiarm spirals emit waves of electrical activity at rates faster than for single-arm spirals and exhibit two distinct behaviors, namely "arm-switching" and "tip-switching." The phenomenon of rate acceleration due to an increase in the number of spiral arms possibly may underlie the acceleration of functional reentrant tachycardias paced by a clinician or an antitachycardia device.

Animals↗

PubMed2004

Ionic currents involved in shock-induced nonlinear changes in transmembrane potential responses of single cardiac cells.

An exhaustive characterization of how an isolated cardiac cell responds to applied electric fields could serve as an important groundwork for understanding responses of more complex higher order systems. Field stimulation of single cardiac cells during the early plateau of the action potential results in a nonuniform change in transmembrane potential (Vm) across the cell length that is more heavily weighted in the negative direction. These negatively shifted Vm responses are not replicated theoretically using present day membrane models. The goal of this study was to explore the membrane currents involved in the field responses during the plateau by selectively blocking various ion channels. Enzymatically isolated single guinea pig cells were stimulated with uniform field S1-S2 pulses, and the transmembrane potential responses were optically recorded from several sites along the cell length to assess the drug effect. We used nine different pharmacological agents to manipulate the conductance of major cardiac ion channels of which only barium (Ba2+) altered the transmembrane potential responses. At 50 microM Ba2+, which specifically blocks inwardly rectifying current I(K1), the negative shift in Vm responses was accentuated. At 1 mM Ba2+ , which blocks both I(K1) and sustained plateau current I(Kp), the negative shift diminished. However, 1 mM Ba2+ also depolarized the cells, and depressed or completely eliminated the action potential. Based on these results we conclude that I(K1) contributes to field-induced responses during the plateau stimulation by passing a net inward current, which when blocked accentuates the negative shift in the Vm responses. A conclusive role of I(Kp) could not be demonstrated because of confounding changes in membrane potential. However, from our results it remains as the most viable candidate for the elusive current that contributes a net outward current to produce negatively weighted Vm responses during plateau stimulation and warrants further investigation.

Animals↗

PubMed2004

Fluorescence imaging of electrical activity in cardiac cells using an all-solid-state system.

Tracking spatial and temporal determinants of cardiac arrhythmogenesis at the cellular level presents challenges to the optical mapping techniques employed. In this paper, we describe a compact system combining two nontraditional low-cost solutions for excitation light sources and emission filters in fluorescence measurements of transmembrane potentials, Vm, or intracellular calcium, [Ca2+]i in cardiac cell networks. This is the first reported use of high-power blue and green light emitting diodes (LEDs), to excite cell monolayers stained with Vm - (di-8-ANEPPS) or [Ca2+]i - (Fluo-3) sensitive dyes. In addition, we use simple techniques for fabrication of suitable thin emission filters with uniform properties, no auto-fluorescence, high durability and good flexibility for imaging Vm or [Ca2+]i. The battery-operated LEDs and the fabricated emission filters, integrated with a fiber-optic system for contact fluorescence imaging, were used as tools to characterize conduction velocity restitution at the macro-scale. The versatility of the LEDs for illumination is further emphasized through 1) demonstration of their usage for epi-illumination recordings at the single-cell level, and 2) demonstration of their unique high-frequency light modulation ability. The LEDs showed excellent stability as excitation light sources for fluorescence measurements; acceptable signal-to-noise ratio and negligible cell photodamage and indicator dye photobleaching were observed.

Action Potentials↗

PubMed2003

Functional reentry in cultured monolayers of neonatal rat cardiac cells.

Previous studies of reentrant arrhythmias in the heart have been performed in computer models and tissue experiments. We hypothesized that confluent monolayers of cardiac cells can provide a simple, controlled, and reproducible experimental model of reentry. Neonatal rat ventricular cells were cultured on 22-mm-diameter coverslips and stained with the voltage-sensitive dye RH-237. Recordings of transmembrane potentials were obtained from 61 sites with the use of a contact fluorescence imaging system. An electrical field stimulus, followed by a point stimulus, induced 39 episodes of sustained reentry and 21 episodes of nonsustained reentry. Sustained reentry consisted of single-loop (n = 18 monolayers) or figure-of-eight (n = 4) patterns. The cycle length, action potential duration at 80% repolarization, and conduction velocity were (in means +/- SE) 358 +/- 33 ms, 118 +/- 12 ms, and 12.9 +/- 1.0 cm/s for single loop and 311 +/- 78 ms, 137 +/- 18 ms, and 7.8 +/- 1.3 cm/s for figure-of-eight, respectively. Electrical termination by 6- to 13-V/cm field pulses or 15- to 20-V point stimuli was successful in 60% of the attempts. In summary, highly stable reentry can be induced, sustained for extensive periods of time, and electrically terminated in monolayers of cultured neonatal rat cardiac myocytes.

Action Potentials↗

PubMed2002

Spatial heterogeneity of transmembrane potential responses of single guinea-pig cardiac cells during electric field stimulation.

Changes in transmembrane voltage (V(m)) of cardiac cells during electric field stimulation have a complex spatial- and time-dependent behaviour that differs significantly from electrical stimulation of space-clamped membranes by current pulses. A multisite optical mapping system was used to obtain 17 or 25 microm resolution maps of V(m) along the long axis of guinea-pig ventricular cells (n = 57) stained with voltage-sensitive dye (di-8-ANEPPS) and stimulated longitudinally with uniform electric field (2, 5 or 10 ms, 3-62 V cm(-1)) pulses (n = 201). The initial polarizations of V(m) responses (V(mr)) varied linearly along the cell length and reversed symmetrically upon field reversal. The remainder of the V(m) responses had parallel time courses among the recording sites, revealing a common time-varying signal component (V(ms)). V(ms) was depolarizing for pulses during rest and hyperpolarizing for pulses during the early plateau phase. V(ms) varied in amplitude and time course with increasing pulse amplitude. Four types of plateau response were observed, with transition points between the different responses occurring when the maximum polarization at the ends of the cell reached values estimated as 60, 110 and 220 mV. Among the cells that had a polarization change of > 200 mV at their ends (for fields > 45 V cm(-1)), some (n = 17/25) had non-parallel time courses among V(m) recordings of the various sites. This implied development of an intracellular field (E(i)) that was found to increase exponentially with time (tau = 7.2 +/- 3.2 ms). Theoretical considerations suggest that V(ms) represents the intracellular potential (phi(i)) as well as the average polarization of the cell, and that V(mr) is the manifestation of the extracellular potential gradient resulting from the field stimulus. For cells undergoing field stimulation, phi(i) acts as the cellular physiological state variable and substitutes for V(m), which is the customary variable for space-clamped membranes.

Animals↗

PubMed2002

Decomposition of field-induced transmembrane potential responses of single cardiac cells.

In this study, we used a multi-site optical mapping system to record field-induced responses of single cells isolated from guinea pig hearts. The cells were stained with voltage sensitive dye di-8-ANEPPS and stimulated with two uniform field (S1-S2) pulses along their longitudinal axes. The first pulse (S1 = 5 ms, <10 V/cm) was applied during rest and elicited an action potential. The second pulse (S2 = 10 ms, 4-50 V/cm) was applied 15 ms after the break of the S1 pulse (during the action potential plateau). The transmembrane potential responses, Vm(F)s, were optically recorded from up to 12 sites along the cell length using a fiber optic based optical mapping system at a resolution of 17 or 25 microm. The field-induced Vm(F)s had a complex spatio-temporal pattern. We show that these responses can be decomposed into simpler components. The first component, termed the differential-mode component (Vmd(F)), is like the response of a passive cell. The second component, termed the common-mode component (Vmc(F)), is identical all along the cell and adds a constant offset to the differential mode response of various sites along the cell length, to produce the total Vm(F) responses of the cell.

Algorithms↗

PubMed2002

A novel algorithm for cardiac biosignal filtering based on filtered residue method.

In this paper, a new algorithm is presented for the filtering (de-noising) of cardiac bioelectrical signals. The primary target of this algorithm is the class of cardiac action potentials recorded using voltage-sensitive dyes, although the method is also applied to electrocardiographic signals. High periodicity is one of the main features of cardiac biosignals. The proposed algorithm exploits this feature in filtering signals with a minimum amount of distortion. The basic idea is to use signal averaging in time to find the stationary portion of the signal. The residue is found by subtracting the signal average from the corresponding points of the input. After passing through a low-pass filter, the filtered residue (FR) is added back to the signal average to reconstruct the output. The practical implementation of the filter residue algorithm is discussed. Stretching and shrinking operations are the basis for the conversion of quasi-periodic signals into periodic signals, which can then be subjected to the FR algorithm. Various examples are presented, and error estimation is performed to guide the selection of optimal parameters for the algorithm. The ability of the algorithm to reconstruct the variation among beats is demonstrated, and its limitations are discussed.

Action Potentials↗

PubMed2002

Inter-pulse interval between rectangular voltage pulses affects electroporation threshold of artificial lipid bilayers.

This paper describes experiments that determine how the inter-pulse interval between rectangular pulses in a train of pulses alters the threshold of electroporation of 1-pamitoyl 2-oleoyl phosphatidycholine bilayer lipid membranes. The bilayers were exposed to a train of sixteen 100-micros duration pulses. Threshold voltage and the sequence number of the pulse in the train, where onset of the electroporation occurred, were recorded for six inter-pulse intervals (infinity, 1000 micros, 100 micros, 10 micros, 1 micros, 0 micros). The threshold voltage of the pulse train decreased linearly with the logarithm of the inter-pulse interval. When the inter-pulse interval was 1 microm, electroporation threshold dropped to that of a single pulse with duration 1600 micros (equal to the sum of all pulse durations in the train). In this case, the occurrence of bilayer rupture was almost equally frequent for all pulses in the train. When the inter-pulse interval between the pulses exceeded 1 micros, the influence of the previous pulse on the response to the following pulse declined. It became more likely that the bilayer ruptured during the first half of the train. These experimental observations suggest that a train of pulses applied with short inter-pulse interval (less than 1 ms) can lower the electroporation threshold of bilayer lipid membranes.

Dose-Response Relationship, Drug↗

PubMed2002

Effects of uniform electric fields on intracellular calcium transients in single cardiac cells.

Although intracellular calcium ([Ca(2+)](i)) transients in cardiac cells have been well studied in the uniformly polarized cell membrane, how these transients are modified during field stimulation when the cell membrane is nonuniformly polarized has not been investigated. In this study we characterized the effects of uniform field stimuli on [Ca(2+)](i) transients in isolated guinea pig cardiac cells. Single guinea pig cells were enzymatically isolated, loaded with the [Ca(2+)](i) fluorescent indicator fluo-3, and stimulated along their longitudinal axes with S1 or S1-S2 (S1-S2 = 50 ms) pulses. The fluorescence signals were recorded simultaneously from up to 12 sites along the cell length using a multisite mapping system. S1 pulse, applied during the resting phase of the action potential, induced [Ca(2+)](i) transients that had an earlier onset at the anodal-facing end, suggesting that [Ca(2+)](i) gradients (nabla[Ca(2+)](i)) develop during the rising phase of the [Ca(2+)](i) transients. With the assumption that the peak change in [Ca(2+)](i) is 980 nM, nabla[Ca(2+)](i) was estimated to be approximately 3.4 nM/microm in the anodal half of the cell for a nominal 10 V/cm field and negligible in the cathodal half. The S2 pulse that was applied during the plateau of the action potential also perturbed the [Ca(2+)](i) transients and produced [Ca(2+)](i) gradients directed from the center to either end of the cell. Mean nabla[Ca(2+)](i) in the anodal half of the cell (approximately 4.2 nM/microm) was found to be statistically higher than in the cathodal half (approximately 2.8 nM/microm).

Animals↗