Andrew E. Pollard

Conduction between isolated rabbit Purkinje and ventricular myocytes coupled by a variable resistance

Conduction at the Purkinje-ventricular junction (PVJ) demonstrates unidirectional block under both physiological and pathophysiological conditions. Although this block is typically attributed to multidimensional electrotonic interactions, we examined possible membrane-level contributions using single, isolated rabbit Purkinje (P) and ventricular (V) myocytes coupled by an electronic circuit. When we varied the junctional resistance ( R j) between paired V myocytes, conduction block occurred at lower R j values during conduction from the smaller to larger myocyte (115 ± 59 MΩ) than from the larger to smaller myocyte (201 ± 51 MΩ). In Purkinje-ventricular myocyte pairs, however, block occurred at lower R j values during P-to-V conduction (85 ± 39 MΩ) than during V-to-P conduction (912 ± 175 MΩ), although there was little difference in the mean cell size. Companion computer simulations, performed to examine how the early plateau currents affected conduction, showed that P-to-V block occurred at lower R j values when the transient outward current was increased or the calcium current was decreased in the model P cell. These results suggest that intrinsic differences in phase 1 repolarization can contribute to unidirectional block at the PVJ.Conduction at the Purkinje-ventricular junction (PVJ) demonstrates unidirectional block under both physiological and pathophysiological conditions. Although this block is typically attributed to multidimensional electrotonic interactions, we examined possible membrane-level contributions using single, isolated rabbit Purkinje (P) and ventricular (V) myocytes coupled by an electronic circuit. When we varied the junctional resistance (Rj) between paired V myocytes, conduction block occurred at lower Rj values during conduction from the smaller to larger myocyte (115 +/- 59 M omega) than from the larger to smaller myocyte (201 +/- 51 M omega). In Purkinje-ventricular myocyte pairs, however, block occurred at lower Rj values during P-to-V conduction (85 +/- 39 M omega) than during V-to-P conduction (912 +/- 175 M omega), although there was little difference in the mean cell size. Companion computer simulations, performed to examine how the early platea currents affected conduction, showed that P-to-V block occurred at lower Rj values when the transient outward current was increased or the calcium current was decreased in the model P cell. These results suggest that intrinsic differences in phase 1 repolarization can contribute to unidirectional block at the PVJ.

Effects of Electrical Shocks on Cai2+ and Vm in Myocyte Cultures

Changes in intracellular calcium concentration (ΔCai2+) induced by electrical shocks may play an important role in defibrillation, but high-resolution ΔCai2+ measurements in a multicellular cardiac tissue and their relationship to corresponding Vm changes (ΔVm) are lacking. Here, we measured shock-induced ΔCai2+ and ΔVm in geometrically defined myocyte cultures. Cell strands (width=0.8 mm) were double-stained with Vm-sensitive dye RH-237 and a low-affinity Cai2+-sensitive dye Fluo-4FF. Shocks (E≈5 to 40 V/cm) were applied during the action potential plateau. Shocks caused transient Cai2+ decrease at sites of both negative and positive ΔVm. Similar Cai2+ changes were observed in an ionic model of adult rat myocytes. Simulations showed that the Cai2+ decrease at sites of ΔV+m was caused by the outward flow of ICaL and troponin binding; at sites of ΔV−m it was caused by inactivation of ICaL combined with extrusion by Na–Ca exchanger and troponin binding. The important role of ICaL was supported by experiments in which application of nifedipine eliminated Cai2+ decrease at ΔV+m sites. Largest ΔCai2+ were observed during shocks of ≈10 V/cm causing simple monophasic ΔVm. Shocks stronger than ≈20 V/cm caused smaller ΔCai2+ and postshock elevation of diastolic ΔCai2+ This was paralleled with occurrence of biphasic negative ΔVm that indicated membrane electroporation. Thus, these data indicate that shocks transiently decrease Cai2+ at sites of both ΔV−m and ΔV+m. Outward flow of ICaL plays an important role in Cai2+ decrease in the ΔV+m areas. Very strong shocks caused smaller negative ΔCai2+ and postshock elevation of diastolic Cai2+, likely caused by membrane electroporation.

Spatial Changes in the Transmembrane Potential During Extracellular Electric Stimulation

The purpose of this study was to determine the spatial changes in the transmembrane potential caused by extracellular electric field stimulation. The transmembrane potential was recorded in 10 guinea pig papillary muscles in a tissue bath using a double-barrel microelectrode. After 20 S1 stimuli, a 10-ms square wave S2 shock field with a 30-ms S1-S2 coupling interval was given via patch shock electrodes 1 cm on either side of the tissue during the action potential plateau. Two shock strengths (2.1+/-0.2 and 6.5+/-0.6 V/cm) were tested with both shock polarities. The recording site was moved across the tissue along fibers with either 200 micrometer (macroscopic group [n=5], 12 consecutive recording sites over a 2. 2-mm tissue length in each muscle) or 20 micrometer (microscopic group [n=5], 21 consecutive recording sites over a 0.4-mm tissue length in each muscle) between adjacent recording sites. In the macroscopic group, the portion of the tissue toward the anode was hyperpolarized, whereas the portion toward the cathode was depolarized, with 1 zero-potential crossing from hyperpolarization to depolarization present near the center of the tissue. In the microscopic group, only 1 zero-potential crossing was observed in the center region of the tissue, whereas, away from the center, only hyperpolarization was observed toward the anode and depolarization toward the cathode. Although these results are consistent with predictions from field stimulation of continuous representations of myocardial structure, ie, the bidomain and cable equation models, they are not consistent with the prediction of depolarization-hyperpolarization oscillation from representations based on cellular-level resistive discontinuities associated with gap junctions, ie, the sawtooth model.

Myocardial Discontinuities A Substrate for Producing Virtual Electrodes That Directly Excite the Myocardium by Shocks

BACKGROUND Theoretical models suggest that an electrical stimulus causes regions of depolarization and hyperpolarization on either side of a myocardial discontinuity. This study determined experimentally whether an artificial discontinuity gives rise to an activation front in response to an electrical stimulus, consistent with the creation of such polarized regions. METHODS AND RESULTS After a thoracotomy in six dogs, a 504-unipolar-electrode plaque was sutured to the right ventricular epicardium to map activations. From a line electrode parallel to one side of the plaque, 10 S1 stimuli were delivered, followed by S2 and S3 stimuli (S1S1, S1S2, S2S3 interval=300 ms). S1 and S3 stimuli were 25 mA; 5-ms S2 stimuli of both polarities were initially 25 mA and increased in 25 mA increments. The plaque was removed, and a transmural incision was made through the ventricular wall in the middle of the mapped region and sutured closed. The plaque was replaced and the stimulation protocol repeated. Before the incision, S2 stimuli directly activated tissue only near the stimulation site. An activation front arose at the border of the directly activated region and propagated across the plaque. As the S2 stimulus strength was increased, the size of the directly activated region increased. After the incision, sufficiently large S2 stimuli caused direct activation of tissue adjacent to the transmural incision as well as at the stimulation site. Activation fronts that arose adjacent to the transmural incision either propagated proximally toward the stimulation site and collided with the activation front originating from the stimulation wire or propagated distally away from the incision. Minimum S2 stimulus strengths activating areas adjacent to the incision were only 45+/-14% (cathode) and 39+/-18% (anode) of the strengths required to directly activate the same area before the incision was formed (P<.05). CONCLUSIONS Myocardial discontinuities can give rise to activation fronts after a stimulus, suggesting the presence of polarized regions adjacent to the discontinuity.

A model study of intramural dispersion of action potential duration in the canine pulmonary conus.

AbstractRegional gradients of action potential duration (APD) due to electrophysiological differences between endocardial, midmyocardial, and epicardial myocytes may exist across the ventricular wall. In addition, activation sequence-induced gradients of APD may occur if intramural fiber rotation accelerates or decelerates the depolarization wave front. To investigate relative contributions of regional and activation sequence-induced gradients to intramural APD dispersion, we simulated action potential propagation in two-dimensional models with idealized geometries representing the canine pulmonary conus. Ionic currents for endocardial myocytes were described using the Luo–Rudy membrane equations. Modifications to IKs approximated action potentials of epicardial and midmyocardial cells. Spatial coupling was modeled with a bidomain representation of tissue structure that included unequal anisotropic conductivity ratios. Activation sequence-induced gradients reached 69 ms cm−1 during a nonuniform activation sequence where the change in orientation between endocardial and epicardial fibers accelerated the depolarization wave front. Regional gradients reached 133 ms cm−1 at the boundary between endocardial and midmyocardial cells. When regional and activation sequence-induced gradients were oriented in opposite directions, overall APD dispersion decreased. When the gradients were oriented in the same direction, overall dispersion measured as high as 202 ms cm−1. This gradient exceeded values previously estimated as sufficient to induce cardiac arrhythmia during premature stimulation and suggests that regional and activation sequence-induced gradients increase arrhythmia vulnerability in the presence of other arrhythmogenic conditions.

Modulation of repolarization in rabbit Purkinje and ventricular myocytes coupled by a variable resistance

Purkinje-ventricular junctions (PVJs) have been implicated as potential sites of arrhythmogenesis, in part because of the dispersion of action potential duration (APD) between Purkinje (P) and ventricular (V) myocytes. To characterize electrotonic modulation of APD as a function of junctional resistance ( R j), we coupled single isolated rabbit P and V myocytes with an electronic circuit. In seven of eight PV myocyte pairs, both APDs shortened on coupling at R j = 50 MΩ. This was in contrast to modulation of APD in paired ventricular myocytes, which demonstrated APD shortening of the intrinsically longer action potential and APD prolongation of the intrinsically shorter action potential. Companion computer simulations, performed to suggest possible mechanisms for the paradoxical shortening of the V action potential in paired P and V myocytes, showed that the difference in intrinsic peak plateau potentials ( V pp) of the P and V myocytes determined whether the V action potential shortened or prolonged on coupling. This difference in V pp caused a large, repolarizing coupling current to flow to the V myocyte, contributing to early inactivation of the L-type calcium current and early activation of the inward rectifier current. These results suggest that intrinsic differences in phase 1 repolarization could yield differing patterns of APD shortening or prolongation in the network of subendocardial PVJs, leaving some PVJs vulnerable to conduction of premature stimuli while other PVJs remain refractory.Purkinje-ventricular junctions (PVJs) have been implicated as potential sites of arrhythmogenesis, in part because of the dispersion of action potential duration (APD) between Purkinje (P) and ventricular (V) myocytes. To characterize electrotonic modulation of APD as a function of junctional resistance (Rj), we coupled single isolated rabbit P and V myocytes with an electronic circuit. In seven of eight PV myocyte pairs, both APDs shortened on coupling at Rj = 50 MOmega. This was in contrast to modulation of APD in paired ventricular myocytes, which demonstrated APD shortening of the intrinsically longer action potential and APD prolongation of the intrinsically shorter action potential. Companion computer simulations, performed to suggest possible mechanisms for the paradoxical shortening of the V action potential in paired P and V myocytes, showed that the difference in intrinsic peak plateau potentials (Vpp) of the P and V myocytes determined whether the V action potential shortened or prolonged on coupling. This difference in Vpp caused a large, repolarizing coupling current to flow to the V myocyte, contributing to early inactivation of the L-type calcium current and early activation of the inward rectifier current. These results suggest that intrinsic differences in phase 1 repolarization could yield differing patterns of APD shortening or prolongation in the network of subendocardial PVJs, leaving some PVJs vulnerable to conduction of premature stimuli while other PVJs remain refractory.

A Novel Approach to Dual Excitation Ratiometric Optical Mapping of Cardiac Action Potentials With Di-4-ANEPPS Using Pulsed LED Excitation

We developed a new method for ratiometric optical mapping of transmembrane potential (Vm) in cardiac preparations stained with di-4-ANEPPS. Vm -dependent shifts of excitation and emission spectra establish two excitation bands (<;481 and >;481 nm) that produce fluorescence changes of opposite polarity within a single emission band (575-620 nm). The ratio of these positive and negative fluorescence signals (excitation ratiometry) increases Vm sensitivity and removes artifacts common to both signals. We pulsed blue (450 ± 10 nm) and cyan (505 ± 15 nm) light emitting diodes (LEDs) at 375 Hz in alternating phase synchronized to a camera (750 frames-per-second). Fluorescence was bandpass filtered (585 ± 20 nm). This produced signals with upright (blue) and inverted (cyan) action potentials (APs) interleaved in sequential frames. In four whole swine hearts with motion chemically arrested, fractional fluorescence for blue, cyan, and ratio signals was 1.2 ± ± 0.3, 1.2 ± 0.3, and 2.4 ± 0.6, respectively. Signal-to-noise ratios were 4.3 ± 1.4, 4.0 ± 1.2, and 5.8 ± 1.9, respectively. After washing out the electromechanical uncoupling agent, we characterized motion artifact by cross-correlating blue, cyan, and ratio signals with a signal with normal AP morphology. Ratiometry improved cross-correlation coefficients from 0.50 ± 0.48 to 0.81 ± 0.25, but did not cancel all motion artifacts. These findings demonstrate the feasibility of pulsed LED excitation ratiometry in myocardium.

Transient outward current modulates discontinuous conduction in rabbit ventricular cell pairs

OBJECTIVE While several studies have demonstrated that the L-type calcium current maintains discontinuous conduction, the contribution of the transient outward current (I(to)) to conduction remains unclear. This study evaluated the effects of I(to) inhibition on conduction between ventricular myocytes. METHODS An electronic circuit with a variable resistance (R(j)) was used to electrically couple single epicardial myocytes isolated from rabbit right ventricle. We inhibited I(to) with 4-aminopyridine superfusion, rate-acceleration, or premature stimulation to evaluate the subsequent effects on conduction delay and the critical R(j), which was quantified as the highest R(j) that could be imposed before conduction failed. RESULTS I(to) inhibition significantly enhanced conduction in all cell pairs (n=23). Pharmacologic inhibition of I(to) resulted in a 32+/-5% decrease in conduction delay and a 36+/-7% increase in critical R(j). Similarly, reduction of the basic cycle length from 2 to 0.5 s resulted in a 31+/-3% decrease in conduction delay and a 31+/-3% increase in critical R(j). Finally, premature action potentials conducted with a 41+/-4% shorter conduction delay and a 73+/-24% higher critical R(j) than basic action potentials. CONCLUSIONS I(to) inhibition significantly enhanced conduction across high R(j). These results suggest I(to) may contribute to rate-dependent conduction abnormalities.

Comparison of Conventional and Biventricular Antitachycardia Pacing in a Geometrically Realistic Model of the Rabbit Ventricle

Introduction: ICDs often are programmed with antitachycardia pacing (ATP) as the first response to ventricular tachycardia (VT). Many ICDs have an additional lead available for ventricular pacing. We hypothesized that using the additional lead for ATP would improve therapy by advancing the orthodromic wavefront, thereby reducing the size of the excitable gap and inducing block of all reentrant activity.

A biophysical model for cardiac microimpedance measurements

Alterations to cell-to-cell electrical conductance and to the structural arrangement of the collagen network in cardiac tissue are recognized contributors to arrhythmia development, yet no present method allows direct in vivo measurements of these conductances at their true microscopic scale. The present report documents such a plan, which involves interstitial multisite stimulation at a subcellular to cellular size scale, and verifies the performance of the method through biophysical modeling. Although elements of the plan have been analyzed previously, their performance as a whole is considered here in a comprehensive way. Our analyses take advantage of a three-dimensional structural framework in which interstitial, intracellular, and membrane components are coupled to one another on the fine size scale, and electrodes are separated from one another as in arrays we fabricate routinely. With this arrangement, determination of passive tissue resistances can be made from measurements taken on top of the currents flowing in active tissue. In particular, our results show that measurements taken at multiple frequencies and electrode separations provide powerful predictions of the underlying tissue resistances in all geometric dimensions. Because of the small electrode size, separation of interstitial from intracellular compartment contributions is readily achieved.