Copyright © 2005, European Society of Cardiology
Acceleration of functional reentry by rapid pacing in anisotropic cardiac monolayers: Formation of multi-wave functional reentries
aDepartment of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, 21205, USA
bDepartment of Biomedical Engineering, Duke University, 3000 Science Drive, Durham, NC, 27708, USA
* Corresponding author. Tel.: +1 919 660 5510; fax: +1 919 684 4488. Email address: nbursac{at}duke.edu
Received 17 June 2005; revised 31 August 2005; accepted 12 September 2005
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Abstract |
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 Top Abstract 1. Introduction2. Methods3. Results4. DiscussionReferences |
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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.
KEYWORDS Cardiac electrophysiology; Optical mapping; Cell culture; Functional reentry; Rate acceleration
See Editorial by I.R. Efimov and C.M. Ripplinger (pages 307–308) in this issue.
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1. Introduction |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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Termination of monomorphic ventricular tachycardia in the clinical setting or with implantable cardioverter defibrillators (ICDs) is commonly attempted by pacing from a single site in the heart at a rate slightly higher than the rate of VT [1]. Besides entrainment and termination, antitachycardia pacing can result in an accelerated rate of the ECG, transition to a rapid monomorphic or polymorphic ECG, and/or induction of ventricular fibrillation [2,3]. Similarly, rapid focal activity in pulmonary vein myocardial sleeves during sinus rhythm, and possibly atrial tachycardia or flutter, can result in formation of sustained wavebreaks and yield atrial fibrillation [4].
There has been only a handful of studies that addressed mechanisms of pacing-induced acceleration of anatomical (i.e. electrical wave rotating around an anatomical obstacle) or functional (i.e. wave rotating around a functional line of block) reentrant tachycardias. In theory, a stationary reentrant wave can be accelerated by two distinct modes: 1) non-multiplication, where a single rotating wave persists but with accelerated dynamics, or 2) multiplication, where a single wave becomes several stable coexisting waves that activate tissue at an accelerated rate. Experimentally, the non-multiplication mode of acceleration was observed in functional reentry due to a pacing-induced shift in the core location [5,6], or cromakalim-induced decrease in the core size [7]. The multiplication mode of acceleration in anatomical reentry involved pacing-induced formation of a "double-wave" reentry (i.e. two waves that rotate at the same direction around a relatively large common obstacle) [8,9]. The only demonstrated acceleration of a functional reentry by multiplication involved the pacing-induced addition of a counter-rotating reentrant wave, i.e. the formation of "figure-of-eight" reentry [5].
In our previous studies, we combined micropatterned growth of neonatal rat cardiomyocytes and optical mapping of transmembrane potentials at a macroscopic (centimeter-size) scale [10] to demonstrate that anatomical reentry in the cardiac cell culture (monolayer) can be initiated and terminated electrically and is associated with an excitable gap [10], that stationary functional reentry can also be initiated and terminated electrically in isotropic monolayers [11], and that methods of microabrasion and microfabrication can be used to control the degree and uniformity of anatomical and functional anisotropy in cultures of neonatal rat cardiomyocytes [12]. In our most recent study [13] we demonstrated that rapid point pacing can convert single loop functional reentry into a stable, accelerated multi-arm reentry, where multiple functional reentrant waves (arms) rotate in the same direction around a common organizing center. In this study we hypothesized that: 1) rapid pacing can stably accelerate functional reentry through formation of various multi-wave reentrant patterns, 2) the number of waves in these patterns is a determinant of the degree of rate acceleration, and 3) the ability to stably accelerate functional reentry is dependent on the shape of the restitution relationships. To test these hypotheses we systematically studied outcomes of rapid pacing during functional reentry in uniformly anisotropic neonatal rat cardiac monolayers, and characterized different spatial patterns and activation rates that resulted from stable wave multiplication or annihilation.
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2. Methods |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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2.1 Cell culture
All animals were treated according to the Institutional Guidelines of Johns Hopkins University School of Medicine and Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication NO. 85-23, revised 1996). Cardiac cells were dissociated from ventricles of 2–3 day old neonatal rats using trypsin and collagenase, and cultured on microabraded 20 mm diameter coverslips to form confluent, uniformly anisotropic cardiac monolayers (velocity anisotropy ratio of 2.1 ± 0.3). Special care was taken to insure full cell confluence and a high degree of structural and functional homogeneity of the monolayers via: a) inspection by a light microscope prior to experiments, b) assessment of conduction patterns during the experiments, and c) immunofluorescent staining for cardiac and non-cardiac cells, and gap junctions after the experiments, as previously described [12]. Monolayers (
15% of total number) with macroscopic structural (e.g. existence of a hole, or a non-cardiac region 
120 µm in size) or functional heterogeneities (e.g. lack of the signal in one of the recording channels, or irregular isochrones during point pacing) were discarded from the present study.
2.2 Experimental protocol
Transmembrane potentials in 5–7 day old cardiac monolayers were recorded using contact fluorescence imaging with the voltage-sensitive dye RH-237, as previously described [10,12,13]. Briefly, 2–10 s episodes of electrical activity were recorded at 61 hexagonally arranged, 2 mm spaced sites at a sampling rate of 1 kHz. Unipolar cathodal point stimuli (1.2 x threshold, 10 ms duration) were applied at a 2 Hz pacing rate in the center of the monolayer using thin platinum wire [12,13]. The pacing rate was increased every minute in steps of 0.5 Hz, and transmembrane potentials were recorded at the end of each step to construct restitution curves (see below). At the supramaximum sustainable pacing rate (i.e. when 1:1 capture was absent in at least 10 recording sites) pacing was stopped while recording was continued. If stable functional reentry did not spontaneously evolve at the end of the pacing, short pacing bursts at 1–1.2 x maximum rate were subsequently applied at the same central site to induce reentry. After functional reentry was induced and equilibrated for 5–10 min, rapid point pacing trains (pulse amplitude <1.4 x threshold to avoid cell damage, pulse duration=10 ms) were applied from one of four peripheral sites (3, 6, 9 or 12 o'clock positions) in an attempt to stably perturb (terminate, accelerate, or decelerate) the reentry. To create different situations present during antitachycardia pacing by ICDs [1–3], pacing was applied relatively far (at least 7 mm) from the reentry core, and the onset time relative to local activation by reentry, the length (3–25 pulses), and the rate (1.1–1.5 x reentry rate) of the pacing train were arbitrarily varied. If reentry was terminated in one of the attempts, rapid pacing was reapplied at the center of the monolayer to induce another functional reentry, and the pacing protocol to perturb the reentry was repeated. If reentry was stably accelerated or decelerated (i.e. new reentry persisted for at least 5 min), pacing trains at 1.1–1.5 x new reentry rate were applied at the same peripheral site to further perturb the reentry. The experiment was ended 45–60 min after the first recording.
2.3 Data analysis
The longitudinal and transverse conduction velocity (CV), action potential duration at 80% repolarization (APD), and wavelength (WL=APD x CV) were measured at sites along the major (longitudinal) and minor (transverse) axes of elliptical wave at least 4 mm away from the pacing site, as previously described [12,13]. The measured parameters were plotted against the pacing period (cycle length, CL), and the resulting plots referred to as "restitution curves" [14].
2.3.1 Restitution analysis
To compare the restitution curves among different monolayers, the parameter value (APD, CV, or WL) at each pacing CL was expressed as a percentage (%) of the value at a basic CL of 500 ms. The obtained normalized restitution curves were fitted with 3rd order polynomials and their ascending portions characterized by measuring: 1) restitution "breadth", defined as the range of pacing CLs on the curve that corresponded to normalized parameter values of 90% or less, and 2) restitution "depth", defined as the range of normalized parameter values between the minimum value on the curve and 90% (Fig. 6A, bottom).
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The same parameters (APD, CV and WL) and CL (i.e. activation period) during functional reentry were averaged for all recording sites in the monolayer that were at least 2 mm away from the reentry cores. The difference between reentry CL and minimum sustainable pacing CL (i.e. the leftmost point on the restitution curve) was measured to assess the room available for rate acceleration (i.e. CL decrease). This difference, denoted
CL, also represents an index of the average temporal excitable gap in the monolayer during reentry.
2.3.2 Phase analysis
Phase analysis was performed as previously described [13,15]. Briefly, voltage signals were low-pass filtered and linearly interpolated to a rectangular mesh with 0.20 x 0.17 mm elements. Phase maps were constructed from the interpolated data using a time delay embedding technique [13,15] with 15 ms time delay. Phase singularity (PS) trajectories and total number of PS (PS#) in the monolayer as a function of time (PS# plot) were used to track the tip motion, chirality (labeled (+) for clockwise and (–) for counterclockwise) and number of transient and stable reentrant waves. Only those PSs that lasted more than 20 ms and were more than 0.5 mm apart were counted for the PS# plots.
2.3.3 Bipolar pseudo-ECG
A global measure of the electrical activity in the cardiac monolayer was assessed in the form of a bipolar pseudo-ECG (pECG), using the method of lead fields, as previously described [11]. Briefly, the pECG was calculated as a potential difference between 2 virtual extracellular electrodes positioned 5 mm above diametrally opposite points of the monolayer, parallel to horizontal edge of the recording hexagonal area [11]. The Fast Fourier Transform (FFT) of pECG was used to characterize the global spatiotemporal periodicity of electrical activity in the monolayer.
2.4 Statistical analysis
Data were expressed as mean ± SD and analyzed using the Student's t test. Correlation and regression analyses were used to evaluate trends in the data sets. Differences were considered to be significant when p<0.05.
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3. Results |
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3.1 Single loop anisotropic functional reentry
Rapid pacing in the center of an anisotropic monolayer readily induced sustained single loop functional reentry (71/84 monolayers, 151 episodes). In a typical induction episode (during the restitution protocol or pacing burst), pacing at a rate of 6.2 ± 1.4 Hz yielded propagation in the wake of the previous wave followed by formation of a single or multiple wavebreaks (i.e. formations of PS pairs) at a distance of 5.1 ± 1.8 mm from the pacing site. After the pacing was stopped all but a single wave annihilated in mutual collisions or against the boundary of the monolayer, resulting in a sustained single loop functional reentry. In the simplest and most common scenario (Fig. 1A), rapid pacing resulted in a single wavebreak and formation of a pair of PSs (symbols "o" in the phase and PS# plots) with opposite chirality (symbols (+) and (–) in the phase plot). Subsequently, one PS drifted and annihilated (symbol "x" in the phase and PS# plots) at the monolayer boundary (or near the initial wavebreak, not shown). Eventually, a stationary (Fig. 1A and B), single loop functional reentry with monomorphic (Fig. 1A) and highly periodic pECG (sharp dominant FFT peak, Fig. 1C) was formed. Different single loop functional reentries (Fig. 1D) exhibited variable rates (5.2 ± 1.7 Hz), different chiralities, morphologically different PS trajectories that were mostly elongated in the direction of anisotropy (double-head arrows), and highly periodic pECGs. In unsuccessful inductions of functional reentry, attempts failed because of development of 2:1 conduction block at the pacing site before the occurrence of a distant wavebreak, annihilation of all formed waves after termination of pacing, or damage of the monolayer underneath the point electrode owing to excessive pacing.
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to +3.2 Acceleration of functional reentry by wave multiplication
In 57/71 monolayers, the burst point pacing always terminated single loop functional reentry, either through direct invasion of the core by pacing-induced waves, or by annihilation of transiently formed wave tips shortly after the pacing was stopped. In the remaining 14/71 monolayers (a relatively low incidence as found for antitachycardia pacing by ICDs [2,3]), the transiently formed wave tips (i.e. transiently increased PS#) evolved after pacing into 2–5 stable reentrant waves (a multi-wave reentry) which activated cells at a rate 10–70% faster than the rate of the preceding single loop reentry. In most of the cases (12/14 monolayers, 44/47 episodes), the accelerated multi-wave reentry consisted of a constant number of relatively stationary waves (i.e. constant PS# with PS trajectories confined to small regions) that rotated at the same frequency and maintained a constant phase relationship relative to each other. These waves activated the entire monolayer at the same average rate, and yielded a monomorphic pECG with a single dominant peak in the FFT spectrum. The most common mode of monomorphic acceleration (11/14 monolayers, 21/47 episodes) was formation of a 2-arm reentry [13,16,17] which consisted of 2 co-rotating waves (i.e. 2 PSs with the same chirality) that maintained a 180° circumferential separation of arms in the periphery (Fig. 2). In this example, the co-rotating waves had stationary and spatially separated PS (tip) trajectories, and exhibited persistent "arm switching", as previously described [13,17]. The rate acceleration of the pECG was evident from the shift of dominant FFT peak to a higher frequency.
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In a few cases (2/14 monolayers, 3/47 episodes), the resulting PS# was not constant with time, but varied around a steady number (e.g. around 5 in Fig. 3A, top right), yielding a polymorphic pECG (Fig. 3A, middle right) and multiple frequency peaks in the FFT spectrum (Fig. 3A, bottom right). The polymorphic nature of the pECG was brought about by the existence of two large, spatially distinct domains (Fig. 3B), each containing one or more stationary reentrant waves (not shown). These waves activated the domains at 2 distinct average rates that were an integer ratio of each other (i.e.
5:4 in the example in Fig. 3B). The border zone between the domains [18] with continuously forming and annihilating wavelets was the source of the variation in the PS#. The two dominant peaks in the FFT spectrum corresponded to the average activation rates in the two domains, while the other peaks resulted from the periodic variation of activation rate at the sites near the border zone arising from the Wenckebach-like 5:4 block (Fig. 3C).
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3.3 Classification of accelerated reentries
Examples of different multi-wave reentrant patterns and their corresponding PS trajectories are shown in Fig. 4. Multi-wave reentries were classified according to the number and direction (chirality) of stably rotating waves. For example, single loop reentry (Fig. 1A and D) was assigned type 1/0 for having 1 stable PS of one chirality and 0 of the opposite chirality. Figure-of-eight reentry was assigned type 1/1 since it has 2 stable counter-rotating waves (i.e. with (+) and (–) chirality, Fig. 4A, top left). Two-arm reentry [13] (Figs. 2 and 4
) was similarly assigned type 2/0, quatrefoil reentry [19] type 2/2, etc. As expected owing to the limited area of the cardiac monolayer, functional reentries with larger numbers of waves occurred less frequently (Table in Fig. 4B). Moreover, the increased number of stable rotating waves in a single monolayer yielded a decrease in average CL, APD, CV, and WL (coefficient of correlation r=–0.83, –0.83, –0.82, and –0.85, respectively, p<0.01) (Fig. 4B, bottom). Thus, the more complex the multi-wave functional reentry was, the more frequent, but slower and narrower were the propagating waves in the monolayer.
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3.4 Deceleration and simplification
Once initiated, multi-wave reentries stably persisted until their disruption by external pacing (usually performed 5–30 min after initiation). Rapid point pacing during multi-wave reentry resulted in: 1) no change in morphology and rate of pECG, 2) stable acceleration and multiplication to an even more complex pattern (increased total PS#), or 3) stable deceleration and simplification to a less complex pattern (decreased total PS#, Fig. 5A). The diagram of all pacing-induced transitions among different reentrant patterns is shown in Fig. 5B. In general, a rapid pacing train of appropriate length and rate could eventually simplify multi-wave reentry to a single loop reentry (1/0), or result in direct termination of all activity (0/0) either by pacing-induced wandering and annihilation of rotating waves against the boundary of the monolayer, or by elimination of the excitable gap and annihilation of rotating waves on refractory tissue, similar to studies in canine hearts [20].
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3.5 Restitution analysis
The restitution relationships were compared between monolayers where multi-wave reentries were induced ("inducible"), and those where only single loop reentry and no acceleration could be induced ("non-inducible"). At basic pacing CL of 500 ms (Fig. 6B), the inducible group had longer APDs, slightly but insignificantly lower CVs, and similar WLs compared to the non-inducible group. In both groups, the average WL at basic CL was larger than the monolayer diameter (Fig. 6B). The minimum sustainable pacing CL (Fig. 6A, middle), CL of single loop reentry, and their difference
CL (an index of temporal excitable gap) were higher in inducible than in non-inducible group (Fig. 6C). Since normalized restitution relationships did not differ between the longitudinal and transverse parameters (i.e., anisotropy was independent of CL, Fig. 6A), they were pooled together for the analysis of the breadth and depth of the restitution (Fig. 6A, bottom). Inducible group exhibited broader and deeper CV restitution, similar APD restitution, and consequently broader and deeper WL restitution curves compared to non-inducible group (Fig. 6D). The maximum slopes of APD restitution as a function of diastolic interval were higher in the inducible (0.84 ± 0.06) than non-inducible (0.62 ± 0.13) group. No APD alternans were observed in either of the groups. |
4. Discussion |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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This study demonstrates that short bursts of point stimuli applied during single loop functional reentry in uniformly anisotropic cardiac monolayers can result in the formation of stable multi-wave functional reentries. Although the resulting multiple reentrant waves move at a slower speed than the initial single loop wave (due to propagation in the refractory tails of other waves), each site in the monolayer is activated by more than one wave, yielding an overall acceleration of the activation rate. The exact degree of the resulting rate acceleration is a complex function of the number and chirality of the rotating waves, distance between reentry cores, core motion and proximity to the boundaries, as well as restitution relationships in the monolayer. As found in this study, the higher the number of stable reentrant waves in the monolayer, the higher was the rate acceleration. Furthermore, the formation of stable multi-wave reentries was facilitated by relatively broad and deep conduction velocity restitution and large excitable gap during single loop reentry.
4.1 Structural and functional homogeneity of the monolayers
During the preparation of uniformly anisotropic cardiac monolayers, special care was taken to insure highest degree of structural homogeneity [12]. Importantly, no macroscopic structural differences were observed between the monolayers with inducible multi-wave reentries and those where no wave multiplication and rate acceleration could be induced. Nevertheless, all cardiac cell monolayers (similar to native tissue) exhibit randomly distributed microscopic heterogeneities of anatomic (e.g. small intercellular clefts, non-cardiac cells, locally varying cell size, shape and orientation) and functional (e.g. locally varying membrane ion currents, cell–cell coupling and electrical load) origin. These heterogeneities, although less important with ordinary planar propagation, may significantly interact with the wave when the excitability and wavelength are dynamically reduced by: 1) rapid pacing during rest, single loop, or multi-wave reentry, as suggested by formation of multiple wavebreaks during pacing (i.e. increased PS# in (Figs. 1, 2 and 5
), and 2) curvature at the tip of the reentrant waves [21], as suggested by the variable shape and size of different PS (tip) trajectories in the single monolayer (Figs. 2 and 4).
Although a recent study demonstrated that the highest density of PS locations during ventricular fibrillation often coincided with relatively large anatomic structures (e.g. blood vessels, trabeculae, papillary muscle) [22], the roles that microscopic anatomic and functional heterogeneities play in meandering, anchoring, stability, rotation rate, and excitable gap of a functional reentry are still not elucidated. The ability to reproducibly and independently introduce user-defined anatomical heterogeneities (through cell micropatterning) and/or alter cell function (through gene or drug manipulation) in cardiac monolayers may provide a unique means to systematically study these questions.
4.2 Monomorphic pECG of multi-wave reentries
The predominantly monomorphic nature of the pECG of multi-wave reentries was a consequence of the similar rotational rates and relatively stationary nature of the coexisting rotating waves (Figs. 2 and 4). These waves were evidently mutually entrained, i.e. adjacent waves merged for a period of time along their common pathways, an event that occurred once per rotation and promoted the synchronization of the period and phase among different regions in a multi-wave pattern. In general, the high spatial stability and temporal periodicity of novel multi-wave reentrant patterns in cardiac monolayers were facilitated by the: 1) relative structural and functional homogeneity of the monolayer that enabled similar rotation rates of the reentrant waves, 2) absence of APD alternans, and 3) relatively small size and bounded nature of the monolayer that limited the number of wavebreaks and allowed annihilation of drifting waves at the boundary. In particular, the use of an appropriately sized cardiac medium, as done in this study, is critical to preserve relevance of obtained results to wave dynamics in the heart, especially in studies that involve formation of multiple waves, and/or possible transition from reentrant to fibrillatory (aperiodic) activity.
4.3 Acceleration by wave multiplication and restitution shape
As suggested by existing differences in APD between the two groups (Fig. 6B), the observed restitution differences (Fig. 6) were likely caused by parameters that control the repolarization phase of cardiac action potential (e.g. amplitude and/or kinetics of Ca and K currents, recovery of Na current, etc.). On the other hand, the larger APDs in the inducible group may not have directly affected the induction and acceleration of reentry, since wavelengths (WL=APD x CV) at basic CL (Fig. 6B) and during single loop reentry (not shown) were comparable between the groups. Furthermore, computational studies in FitzHugh–Nagumo type models showed that rate acceleration was only feasible in media with marginally low excitability [23]. In our study, the similar CVs at basic CL in the two groups (Fig. 6B) indicate that excitability (i.e. Na current) at rest was not lower in the inducible vs. non-inducible group. Nevertheless, the differences in CV restitutions (Fig. 6A middle panel) suggest that excitability during rapid pacing and single loop reentry was dynamically decreased (by wavefront propagation in the wake of refractory tissue) to a greater extent in the inducible group. Rate acceleration during multi-wave reentry resulted in a further decrease of excitability.
Importantly, WL at basic CL in our cardiac monolayers was larger than the monolayer size (Fig. 6B). Consequently, for single loop reentry to fit inside the monolayer, WL must decrease, i.e. reentry CL (and thus CL) must lie on the ascending part of the WL restitution curve, as characterized by its breadth and depth (Fig. 6D). Therefore, large CL (i.e. large available room for reentry acceleration) can be supported only if WL restitution is broad. On the other hand, WL (=reentry WL–minimum WL) represents the room available for WL decrease, which is necessary for successful wave multiplication in the monolayer. Analogous to CL, large WL (i.e. increased chance for wave multiplication) can be supported only if WL restitution is deep. Therefore, broad and deep restitutions, as found in the inducible monolayers, facilitate reentry acceleration via the multi-wave mechanism. Further studies are needed to elucidate whether broad and deep CV restitutions in general (i.e. independent of the underlying ionic mechanisms) yield relatively large excitable gaps during single loop functional reentry, and favor reentry acceleration. In a broader context, the main question remains how CL of a spiral wave depends on the APD and CV restitution relationships.
4.4 Clinical implications
Our results suggest that relatively broad and deep CV restitution of cardiac tissue is associated with a higher likelihood of stable acceleration of functional reentry (via a multi-reentrant mechanism) during antitachycardia pacing. Conceivably, this rate acceleration, if sufficiently high, may result in new wavebreaks owing to the abundance of structural or functional heterogeneities in the heart [24], and/or presence of dynamic instability of APD [25] and yield fibrillation.
In addition, similar to findings during fibrillation in sheep ventricles [18], our study describes a Wenckebach-like mechanism of fibrillatory conduction between the two spatial domains with different activation frequencies (Fig. 3). The resulting progressive and periodic activation delays near the domains' boundary introduced FFT peaks at frequencies lower than the two dominant frequencies in the domains (Fig. 3C), suggesting a mechanism that contributes to broadening of the FFT spectra during fibrillation.
4.5 Study limitations
One limitation of this study is that uniformly anisotropic monolayers of neonatal rat ventricular myocytes differ from adult ventricular tissues with respect to cell morphology, membrane electrophysiological properties, gap junction distribution, bounded 2-dimensional geometry, and relatively high degree of structural and functional homogeneity. On the other hand, the average wavelength of a solitary propagating pulse both in our cardiac monolayers and in ventricles is larger than the tissue size. Under these conditions, the shapes of normalized restitution relationships, when similar in monolayers and native cardiac tissues, may play similar roles in the induction and dynamics of functional reentries.
Another limitation is that this study was not designed to systematically assess the effects of different pacing parameters (e.g. site, timings, amplitude, electrode size) on termination of functional reentry. For example, a larger pacing amplitude or electrode size [5,6,20], and/or pacing near the reentry core [6] may terminate the reentry more efficiently, and therefore increase the number of terminated vs. accelerated cases compared with our results. Nevertheless, the mechanisms and characteristics of stable acceleration, as found in this study (e.g. the formation of multi-wave patterns with different activation rates, dependence on the restitution properties), are not expected to change for different pacing parameters.
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Acknowledgements |
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This work was supported by NIH grant HL66239 to L.T. and an American Heart Association, Mid-Atlantic Affiliate fellowship to N.B.
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Notes |
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Time for primary review 21 days
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