Tissue Discontinuities Affect Conduction Velocity Restitution
A Mechanism by Which Structural Barriers May Promote Wave Break
Background— The mechanism by which structural barriers promote wave break and fibrillation is unclear. Conduction velocity (CV) restitution is an important determinant of wave break. Abnormal CV restitution is associated with ventricular fibrillation in patients with heart disease and arises preferentially in fibrotic myocardium. We hypothesize that tissue discontinuities imposed by structural barriers cause abnormal CV restitution.
Methods and Results— Tissue discontinuities were simulated in cultures of neonatal rat heart cells grown in 8-armed star patterns. Premature stimulation was applied at the extremity of 1 arm (n=12) while extracellular electrograms were recorded at 24 sites throughout the star. Action potentials were recorded at the following 3 sites: in the stimulated arm and at the discontinuity both proximal to and distal from the star center. Extracellular recordings revealed progressive increases in activation delay (indicative for abnormal CV restitution) only at the discontinuity from arms proximal to the star center. The mean increase in delay was 0.81±0.41 ms/10 ms for recording sites proximal to and 3.13±0.58 ms/10 ms for sites distal from this discontinuity. Depolarizing currents were determined in single cells during premature stimulation and for voltage configurations similar to those arising at the discontinuity. Both voltage-clamp measurements and computer simulations showed that delay at the discontinuity was associated with biphasic, prolonged activation and delayed inactivation of depolarizing current.
Conclusions— Tissue discontinuities cause abnormal CV restitution. Rapid increase in activation after an initial slow activation and delayed inactivation at the discontinuity lengthen the duration of depolarizing current and cause the abnormal restitution.
Received January 23, 2003; revision received April 17, 2003; accepted April 18, 2003.
The role of structural barriers in promoting wave break and fibrillation is unclear. There is evidence that structural barriers provide anchors for stable reentry rather than promoting wave break and fibrillation.1–3 Other studies, however, suggest that such barriers increase vulnerability for fibrillation.4,5 Action potential duration (APD) restitution and conduction velocity (CV) restitution properties are important determinants of the stability of reentrant arrhythmias and may predict the occurrence of ventricular fibrillation (VF).6–8 Saumarez et al9 reported a progressive increase in conduction delay after premature stimulation, indicative of abnormal CV restitution, in patients with hypertrophic cardiomyopathy and prone to VF.
Using a computer model for action potential propagation in a 2D sheet of simulated cardiac tissue, Qu et al7 showed that a rapid CV restitution imposed by fast inactivation kinetics, as is the case in healthy myocardium, prevented spiral wave breakup. This is in line with observations made in control subjects and patients at low risk for VF, in whom activation delay characteristically remains constant until the coupling interval closely approaches the refractory period.9 In patients prone to VF, activation delay increases progressively, starting at a long coupling interval of the premature stimulus. Recordings made in human hearts explanted in the end stage of heart failure revealed that progressive increases in activation delay occurred preferentially in areas with stringy or patchy fibrosis.10 Discontinuities in myocardial bundles and wave-front curvature around anatomic obstacles cause activation delay as a result of load mismatch at those sites.11–14 We hypothesize that tissue discontinuities cause not only activation delay but also, more importantly, a progressive increase of the delay after premature stimulation, indicative of abnormal CV restitution.
The present study was designed to clarify whether tissue discontinuities give rise to abnormal CV restitution and to delineate the mechanism. To answer these questions, patterned structures of neonatal rat heart cells simulating tissue discontinuities and single-cell recordings and computer modeling were used. The following electrophysiological parameters were determined: (1) conduction properties during premature stimulation, (2) action potential characteristics, (3) amplitude and duration of depolarizing current during premature stimulation, and (4) (in)activation characteristics of depolarizing current.
Patterned Cell Cultures
Neonatal rat heart cells were grown in star-shaped patterns with 8 arms (0.1 mm wide and 1.6 mm long) according to methods developed by Rohr et al.15 Measurements were performed in culture medium at 34°C. Stimulation was performed at the extremity of 1 arm (n=12). Unipolar extracellular electrograms were recorded simultaneously at 24 sites distributed homogeneously over all arms. Electrograms were bandpass-filtered (0.5 Hz to 0.5 kHz), amplified 40 times, and digitized (12-bit resolution) at a sample rate of 2 kHz per channel. The point of steepest negative dV/dt in the electrogram was used as local activation time. Pacing was at twice diastolic current threshold, with an 8-pulse drive train (basic cycle length [BCL] 400 or 500 ms) and 1 premature stimulus. Coupling intervals of premature stimuli were from 300 ms down to the refractory period in steps of 10 ms.
Conduction curves were determined at all 24 recording sites by plotting activation delay (interval between stimulus and activation time) against the coupling interval of the premature stimulus. To characterize conduction curves, we used the mean increase in delay (MID). This parameter was calculated by dividing the integrated increase in delay by the interval between the BCL and the refractory period.10 Action potentials were recorded with conventional microelectrodes. Recording sites were in the stimulated arm (n=13), the entrance of the discontinuity (transition from stimulated arm to star center; n=21), and the exit of the discontinuity (transition from star center to arm; n=5). Tip resistance of the electrodes was typically 30 MΩ. Action potentials were digitized at 2 kHz.
Patch-clamp measurements were performed to determine the depolarizing current of single cells for 2 different voltage protocols. Neonatal rat heart cells were seeded in Petri dishes at 1.5×103 cells/cm2. Before measurements, culture medium was replaced with modified Tyrode’s solution, containing (in mmol/L) NaCl 140, KCl 5.4, MgCl2 1.0, CaCl2 1.8, HEPES 5, and glucose 5. Patch electrodes were backfilled with 0.22 μm filtered solution containing the following (in mmol/L): potassium gluconate 130, KCl 10, MgCl2 10, CaCl2 0.6, EGTA 10 ([free Ca2+]=23.5 nmol/L), Na2ATP 5, and HEPES 10. Typical electrode resistance was 5 MΩ.
Cultured cells in Petri dishes were placed on the heated head stage (34°C) of an inverted microscope. Electrodes were mounted in a micromanipulator and connected to the head stage of an Axoclamp 200B amplifier. After formation of gigaohm seals, the membrane patch under the electrode was broken by gentle suction to obtain the whole-cell configuration. Command potentials and recordings were digitized at 20 kHz/channel.
Propagation of activation at a tissue discontinuity was simulated in a linear strand of 90 cells. We used the human ventricular cell model of Priebe and Beuckelmann16 in a numerical representation of the cable equation, similar to that used by Shaw and Rudy.17 Cytoplasmic resistivity was set to 150 Ω · cm and intercellular coupling conductance to 2 μS. The fast sodium current (INa) was scaled down by a factor of 4 to obtain a maximum upstroke velocity of ≈30 V/s and a CV of ≈30 cm/s in the model strand, similar to what we observed in the cell cultures. A tissue discontinuity was introduced by doubling the number of cells at positions 41 to 50 (see Figure 6D). The strand was stimulated at cell 1 at a BCL of 800 ms. A premature stimulus was delivered after 8 basic stimuli.
Values are expressed as mean ± SEM. Statistical analyses of intracellular recordings were performed by ANOVA and paired or unpaired t tests, using SPSS 10 software. Differences were regarded as statistically significant at a value of P≤0.05.
Conduction Delay After Premature Stimulation
Figure 1 shows conduction curves at 6 recording sites. For recording sites in the paced arm, the MID of the conduction curves was small, at 0.81±0.41 ms/10 ms. MID increased only slightly for sites in the stimulated arm farther away from the stimulus. In the remaining 7 arms, MID was increased significantly, to 3.13±0.58 ms/10 ms. Within these arms, too, MID increased only slightly for more distal sites. Activation delay started to increase at the discontinuity from the paced arm to the star center.
Action Potential Recordings
Preparations were stimulated at the extremity of 1 arm of the star, and recordings were made (1) in the stimulated arm (T in Figure 2), (2) at the entrance of the discontinuity (ED), and (3) at the exit of the discontinuity (ExD). Pacing was done at twice diastolic threshold with an 8-pulse drive train (BCL 400 ms) and 1 premature stimulus (Figure 2, bottom left). The coupling interval of the premature stimulus was reduced in steps of 10 ms starting at 300 ms until conduction block occurred at the star center. This coupling interval of the premature stimulus always exceeded the refractory period at the site of stimulation (mean difference, 35 ms). Action potentials recorded in the tract (Figure 2, top left) and at the exit of the discontinuity (Figure 2, top right) always showed a single upstroke. In contrast, action potentials at the entrance of the discontinuity (Figure 2, bottom right) characteristically revealed a notched upstroke (asterisk) after a premature stimulus.
The following parameters of action potentials were determined for basic and premature stimuli (1) amplitude, (2) maximum upstroke velocity (dV/dt), and (3) APD at 50% of the amplitude (APD50). The tables in Figure 3 show the values of the parameters for the basic stimulus and a premature stimulus 10 ms before conduction block at the entrance of the discontinuity (mean value, 187±7 ms). The action potential amplitude at the entrance and exit of the discontinuity significantly reduced after the premature stimulus. Upstroke velocity decreased significantly in the tract and at the entrance of the discontinuity. APD recorded at the entrance to the discontinuity was not significantly affected by premature stimulation, in contrast to APD recorded at sites in the tract and at the exit of the discontinuity.
Recording the Inward Ionic Current
To assess the effect of premature stimuli on availability of intrinsic inward current, voltage-clamp recordings of single myocytes were performed (n=10; Figure 4). Eight pulses (80 mV; 140 ms duration; holding potential, −70 mV) at 400-ms intervals followed by a premature pulse were applied. The coupling interval of the premature pulse was decreased from 300 to 140 ms. Figure 4A shows superimposed command potentials (top) and resulting current traces (bottom). Peak inward current (asterisk) decreases with decreasing coupling interval of the premature stimulus. Figure 4B shows the ratio of the peak inward current during the premature stimulus (asterisk) and the peak current of the basic stimulus (arrow). The inward current starts to decrease at a coupling interval of the premature stimulus of ≈270 ms. At 180 ms, the decrease is 23±6%, which correlates with the 18±9% decrease in upstroke velocity observed in the tracts. The reduced inward current may account for not only the decreased upstroke velocity but also the reduced APD in the tracts (Figure 3, left columns).
At short coupling intervals of the premature stimulus, the action potential at the entrance of the discontinuity typically reveals a notch (Figure 2, bottom right, asterisk) during which the voltage remained virtually constant for a short duration. To assess the role of this “equipotential phase” in the action potential on the inward current, a voltage-clamp protocol simulating this phase, as illustrated in Figure 5A, was applied to single cells (n=7). Duration of the phase was 2 ms, and the “notch voltage” was increased from −60 to 0 mV in steps of 10 mV. Typical inward currents for 3 different notch voltages are shown in Figure 5B. For voltages of approximately −30 mV, the inward current reveals 2 components (arrows in blue tracing). This indicates that after an initial depolarizing voltage step and a hold potential with a duration of 2 ms, channel availability for inward current is not completely abolished. Depending on the voltage of the notch, additional channels are opened after a second depolarizing step, resulting in the second current peak. Figure 5C shows the mean normalized amplitudes of the 2 peak currents in dependence of the notch voltage.
Activation and Inactivation Parameters
With a peak calcium current to peak INa ratio of 0.05±0.03 (n=5), quite similar to the model value of 0.03, the main component of intrinsic inward current in our cell cultures is the sodium current. Therefore, we further assessed the mechanism underlying the notched upstroke at the entrance of the discontinuity by studying INa and its (in)activation parameters m, h, and j in the computer model.
First, we tested whether our model could reproduce the experimental results. We applied the voltage-clamp protocols of Figures 4A and 5⇑A to a single model cell. The results shown in Figures 4B and 6⇑A and those shown in Figures 5C and 6⇑B are comparable. Figure 6C shows that the computed conduction curves are similar to those obtained experimentally (Figure 1B). Next, the action potential, the inward current INa, its activation parameter (m3), and its fast (h) and slow (j) inactivation parameters were calculated at a site in the first, “normal” segment of the strand (cell 20 of cells 1 to 40) and at the entrance of the discontinuity, where a single cell (cell 40) is connected to the first 2 cells of the middle, “thick” segment of the strand (Figure 6D). Figure 6E shows that a premature stimulus with a coupling interval of 400 ms generates an action potential in cell 20 with a smooth upstroke (red line). In contrast, the action potential at the entrance of the discontinuity is notched, with a short “equipotential phase” (blue line). The inward current INa at the entrance of the discontinuity (Figure 6F, blue line) reveals 2 peaks, similar to those found in the voltage-clamp experiments. Only 1 sharp current peak appears for cell 20 (red line). (In)activation parameters for cell 20 (red lines in Figure 6G) show a smooth but rapid change. The activation parameter m3 of cell 40 (solid blue line), however, starts with a foot during the first depolarizing step and is followed by a fast rise after the second depolarizing step. The course of the parameters h and j shows that inactivation is delayed (dashed and dotted blue lines). The fast increase in m3 after the second depolarizing step, together with delayed inactivation parameters h and j, account for the second peak in the depolarizing current.
Several studies have shown that structural barriers, such as fibrosis, increase susceptibility for wave break and fibrillation.4,5 Other studies, however, suggest that such barriers promote stability by anchoring meandering spiral waves.1,2 In the present study, we show that tissue discontinuities imposed by structural barriers affect CV restitution in favor of wave break.6–8 Abnormal CV restitution is reflected by a progressive increase of activation delay after premature stimulation. Biphasic activation together with delayed inactivation of myocardial cells at tissue discontinuities lengthens the time during which depolarizing current is available and results in abnormal CV restitution.
Structural Barriers and Tissue Discontinuities
Observations that structural barriers stabilize reentrant wave fronts are contradictory to the fact that fibrillation is frequently found in chronically diseased myocardium showing increased deposition of collagen. An explanation for this discrepancy might be that in the studies showing stabilization of spiral waves, barriers were often compact, comprising holes,2 artificial lines of block,1 or cardiac vessels.3 In chronically diseased myocardium, cardiac remodeling is associated with increased collagen deposition exhibiting a complex architecture consisting of an intermingling of collagenous and myocardial fibers.13,18 Myocytes within such areas usually exhibit near-normal intracellular electrophysiological characteristics, but extracellular electrograms are often fractionated13,14,18,19 as a result of discontinuous conduction properties in these areas.4,20 The observations suggest that tissue discontinuities are present in these compromised areas. Propagation of the electrical impulse at several types of discontinuity have been studied (1) at sites with sudden change in fiber diameter, (2) at the extremity of inexcitable barriers where wave fronts curve, and (3) at inexcitable barriers with a narrow isthmus.11,12,20 These studies focused on activation delay; we investigated the role of load mismatch imposed by a sudden change in fiber diameter in CV restitution.
Cardiac Electrical Restitution
Restitution properties of the cardiac action potential and CV have been shown to be important determinants of the stability of reentrant arrhythmias.6–8 In this study, we focused on CV restitution. Qu et al7 showed that CV restitution promotes spiral wave breakup independently of APD restitution. Spiral wave breakup was promoted when the slope of the CV restitution curve was less steep. Similar observations were made by Sampson and Henriquez,21 who showed that a steep CV restitution slope is beneficial in overcoming intrinsic cellular heterogeneity after premature beats. These data are compatible with clinical observations made by Saumarez et al,9 who showed that in patients prone to VF, conduction curves have abnormal characteristics, yielding a progressive increase in conduction delay starting at long coupling intervals of the premature stimulus. In healthy myocardium, conduction delay remains constant until the coupling interval approaches the refractory period and a steep increase in delay ensues.
Ionic Current Flow at Tissue Discontinuities
Antegrade and retrograde impulse conduction at a region of abrupt tissue expansion has been investigated by Fast and Kléber,11 who used cell cultures and computer simulations. They evaluated the critical dimensions of the tissue for establishing unidirectional conduction block. They observed 2 rising phases in the action potential at the discontinuity, similar to our observations. In our study, however, biphasic upstrokes were seen primarily after premature stimulation. This discrepancy is probably because of the more moderate amount of tissue expansion in our study. In contrast to our observations of ionic current flow at the discontinuity, the mathematical modeling of Fast and Kléber11 did not show a second local ionic current peak that coincided with the second rising portion of the upstroke and contributed to the local depolarizing current and the downstream electrotonic current flow. This may be because of the differences in sink-source relationships and the associated electrotonic current flow between the 2 models.
Conduction Delay at Tissue Discontinuity
Results from both the present study and others show that profound conduction delay may occur at tissue discontinuities. The computer simulations in this study show that the amount of charge at the discontinuity (surface beneath the sodium current curve in Figure 6F) is increased by a factor of 1.8 during premature stimulation, which compares with the increased load at the discontinuity (factor of 2.0). This increase in charge is caused primarily by the increase in duration of the sodium current. There was no significant change in duration of the sodium current at cell 20 in the tract during the premature stimulus. The increase of the duration of the sodium current at the discontinuity (cell 40) during the premature stimulus is the most likely explanation for the progressive increase in activation delay after premature stimulation.
Clinical and experimental data suggest an association between conduction abnormalities, fibrosis, and ventricular arrhythmias or sudden death.8,18 Although nonuniform anisotropy in patients with ventricular tachycardias has been shown to be associated with reentry in healed myocardial infarction, it is not known what degree of nonuniformity is needed to alter vulnerability to VF. Our data show that tissue discontinuities may change CV restitution in such a way that spiral wave breakup is promoted, enhancing vulnerability to VF. Such discontinuities may be expected in hearts with structural heart disease.
The present study was performed in a 2D preparation with a fixed discontinuity; the third dimension has been neglected. In addition, discontinuities imposed by the intermingling of surviving myocardial tissue and fibrosis in compromised myocardial tissue will vary, and we cannot be sure about the effect on restitution in the entire heart. We determined the effect of tissue discontinuity on CV restitution by applying premature stimuli, which differs from the conventional method, in which pacing with decremental cycle lengths is used.
This study was supported by the Netherlands Heart Foundation (grant No. 99.200). The authors thank Drs Kléber and Rohr for support in setting up the cell culture technique and Dr Goedbloed and M. van den Boogaart for development and production of the electrode arrays.
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