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Cardiovascular Research Laboratory, Departments of Medicine (Cardiology), Pediatrics (Cardiology), and Physiology, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California
Submitted 23 October 2007 ; accepted in final form 21 January 2008
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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calcium cycling; arrhythmias
Neonatal rat ventricular myocyte monolayers provide a simple two-dimensional (2D) tissue model to explore the relevance of dynamic factors to spatially discordant alternans. Here we used optical mapping of membrane voltage and Cai in monolayers to determine whether spatially discordant alternans could be induced, and, if so, whether its behavior supported a role of dynamic factors. In addition to control conditions, we studied the effects of the L-type Ca current agonist BAY K 8644, which enhances dynamic factors by steepening APD restitution slope and promoting Cai cycling instability by loading the sarcoplasmic reticulum (SR) with high levels of Ca. Our findings provide strong additional experimental support for the role of dynamic factors in the generation of spatially discordant alternans and directly demonstrate that spatially discordant alternans can lead to conduction block and initiation of reentry in a preparation without any unmapped subsurface tissue.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Neonatal rat ventricular myocytes from 2- to 3-day-old Sprague-Dawley rats were obtained by standard methods (24), plated on 22 x 22-mm plastic coverslips (106 myocytes per coverslip), and cultured for 6–7 days. Monolayers with insufficient cellular confluence, as assessed by phase-contrast microscopy, or uneven propagation patterns during pacing at 2 Hz were excluded from analysis. Monolayers were studied under two different conditions: control (n = 37) and during exposure to BAY K 8644 (racemic, 0.1 µM, Sigma, n = 28). The use and care of the animals in these experiments were approved by the Chancellor's Animal Research Committee at the University of California Los Angeles.
Stimulation Protocol
Unipolar point stimuli were delivered at the edge of the coverslip using a Grass stimulator triggered by computer-controlled pacing sequences. Specimens were paced at 2 Hz. Then a rapid pacing protocol was performed, initially at 340-ms cycle length, decremented by 20 ms every eight beats until reaching 140-ms cycle length.
Optical Mapping System
Monolayers were stained by immersion into oxygenated Tyrode's solution (in mM, 136 NaCl, 5.4 KCl, 1.8 CaCl2, 0.33 NaH2PO4, 1 MgCl2, 10 HEPES, and 10 glucose, pH 7.3) at 37°C, containing the fluorescent voltage dye RH-237 (5 µM for 5 min) and/or the Ca dye rhod 2-AM or rhod-FF (5 µM for 40 min) plus 0.016% (wt/wt) pluronic (Molecular Probes, Eugene, OR), and then placed in a perfusion bath. Experiments were conducted at 37°C. Fluorescence was excited by two light sources (each with 4 light-emitting diodes; Luxeon, Ontario, Canada) filtered at 540 ± 20 nm. The emitted fluorescence was separated using a dichroic mirror (at 630 nm), directed to two separate cameras with their corresponding emission filters (715 nm for RH-237 and 585 nm for rhod 2/rhod-FF).
Charge-coupled device camera recordings. We used electron-multiplying, back-illuminated, cooled charge-coupled device (CCD) cameras (Photometrics Cascade 128+), with 128 x 128 spatial resolution at 0.6- to 5-ms per frame. Signals were digitized with 16 bits of precision. Single-dye staining with rhod 2-AM (n = 22) or RH-237 (n = 19) confirmed the absence of cross talk between voltage and Ca signals, and simultaneous voltage and Ca mapping were performed in 15 specimens.
Photodiode array. In nine additional experiments, voltage (RH-237 staining) was also recorded optically using a photodiode array (PDA) (WuTech), consisting of 464 hexagonally arranged sites with 720-µm spacing.
Data Analysis
Raw data were processed with custom-written software. When the goal was to optimize spatial resolution (at the expense of temporal resolution), CCD recordings were subjected to 1) spatial filter (2 x 2 binning); 2) 5-point median temporal filter; 3) polynomial curve fitting to eliminate baseline drift caused by photobleaching; and 4) range normalization. After processing, this yielded a final spatial resolution of 64 x 64 (340 x 340 µm/pixel) and a temporal resolution of 15–25 ms (3–5 ms per frame x 5). When the goal was to optimize temporal resolution (at the expense of spatial resolution), CCD recordings were subjected to spatial filter (4 x 4 binning), and the other same algorithms were applied. This yielded a postprocessing spatial resolution of 32 x 32 (680 x 680 µm/pixel) and a final temporal resolution of 3 ms (0.6 ms per frame x 5). As an alternative method to enhance temporal resolution, we also used PDA recordings in place of CCD recordings in some experiments, with no spatial or temporal filtering applied to the PDA traces.
APD was measured at 80% repolarization (APD80) using custom-written software to detect the time to repolarize to 80% from the peak amplitude of the AP back to the take-off point of the same AP. This may have modestly underestimated the amplitude of APD alternans when the take-off point was also alternating, but did not affect the detection of the onset or offset of APD alternans. We chose APD80 rather than APD at 90% repolarization (APD90) to minimize measurement ambiguity when full repolarization was not achieved at rapid pacing rates. Diastolic interval (DI) was measured from 80% repolarization of the previous AP to the next AP upstroke. Cai transient amplitude was measured as the absolute increase in fluorescence (minimum to peak in arbitrary units). APD and Cai alternans were considered to be present when APD80 and Cai transient amplitude alternated by >10 ms and >10%, respectively, on a beat-to-beat basis, and persisted until the end of the pacing protocol. Difference maps between APD80 and Cai transient amplitude on successive beats were generated and shaded red if the differences were positive, and green if negative. Thus, during spatially discordant alternans, red and green regions alternated, separated by a nonalternating nodal line (white). CV was estimated using voltage signal with custom-written software, which detected the activation time of each pixel and measured the difference in activation time between two points separated by a known distance located orthogonally to propagation direction. To avoid artifacts from direct activation of myocytes near the pacing site (virtual electrode), CV was calculated from the average difference in activation time between a site 5 mm from the pacing site and three equidistant sites as remote as possible from the pacing site. APD80 and CV restitution curves were generated by plotting APD80 or CV vs. DI and fitting to a single exponential. APD restitution slopes were calculated by differentiating the exponential fits using Origin (Microcal) software. Maximum APD restitution slope was obtained by calculating the value of this slope at the shortest DI that elicited an AP. The conduction block at the nodal line was defined as block within 1 mm of the nodal line.
Statistics
Data are shown as means ± SD. Statistical tests included 
2 test and Student's T-test. P values <0.05 were considered significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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We examined 37 monolayers under control conditions, with optical mapping of Cai alone (n = 10), voltage alone (n = 16), or both simultaneously (n = 11). A bright-field image of a representative confluent monolayer meeting our inclusion criteria is shown in Fig. 1A. Figure 1B shows representative optical traces of membrane voltage [voltage fluorescence (FV)] and Cai [Cai fluorescence (FCa)]. APD averaged 126 ± 12 ms, and Cai transient duration (full width at half-maximal amplitude) averaged 174 ± 14 ms. In all monolayers meeting our inclusion criteria, propagation was uniform without conduction block when paced at 2 Hz from either of two orthogonally positioned sites (Fig. 1, C and D). CV averaged 0.24 ± 0.1 m/s in both orthogonal directions.
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180 ms) in control conditions, and 85–68 ms (corresponding to pacing cycle lengths 200 ms) after BAY K 8644 (Fig. 2D). Figure 2C shows that significant CV restitution was present (solid black line), and CV was further reduced by BAY K 8644 at short DIs (red line).
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Pacing-induced Cai Transient and APD Alternans Under Control Conditions
Spatially concordant and discordant alternans. Monolayers could be paced to a cycle length of 156 ± 14 ms before 1:1 capture was lost. Spatially concordant Cai transient alternans was induced in all monolayers, at an average pacing cycle length of 225 ± 16 ms, and became spatially discordant in 13 of 21 specimens (62%) at an average pacing cycle length of 213 ± 16 ms. CV restitution was more prominent in monolayers that transitioned to spatial discordant alternans (Fig. 2C, open squares) than in those that did not (open circles). Figure 3 shows an example of the Cai transient progressing from spatially concordant to discordant alternans. Since Fast et al. (6, 7) reported that the high-Ca affinity of rhod 2-AM can lead to spuriously prolonged Cai transients, we also stained three monolayers with rhod-FF-AM (lower Ca affinity) in place of rhod 2-AM. The incidence or onset of Ca alternans did not differ significantly (data not shown).
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Spatially concordant and discordant alternans. We examined 28 monolayers after exposure to BAY K 8644, mapping either Ca alone (n = 12), voltage alone (n = 12), or both simultaneously (n = 4). After BAY K 8644, 1:1 conduction failed at a longer pacing cycle length of 176 ± 22 ms, compared with 156 ± 14 ms before BAY K 8644 (P < 0.05). Spartially concordant Cai transient and APD alternans were induced in all (16 of 16) monolayers after BAY K 8644, with the onset at an average pacing cycle of 270 ± 16 ms, compared with 225 ± 6 ms in control conditions (P < 0.05). BAY K 8644 also increased the incidence of spatially discordant Cai transient and APD alternans from 62 to 100% (P < 0.01), which occurred at an average pacing cycle length of 244 ± 16 ms, compared with 213 ± 16 ms before BAY K 8644 (P < 0.05). The maximum amplitude of APD alternans was significantly greater (35 ± 6 vs. 18 ± 5 ms in control, P < 0.05), as was the maximum amplitude of Cai transient alternans (53 ± 14 vs. 35 ± 18% in control, P < 0.05).
Nodal line behavior after BAY K 8644. Pacing rate and site had similar effects on nodal line position and orientation after BAY K 8644 (not shown) as under control conditions. BAY K 8644 significantly increased the maximum number of nodal lines. Figure 6 shows an example of multiple APD nodal lines after BAY K 8644. The average number of Cai nodal lines increased from 2.1 ± 0.7 to 3.5 ± 0.3 (P < 0.01; Fig. 6C).), and the average number of APD nodal lines from 2.1 ± 0.4 to 3.1 ± 0.5 (P < 0.01; Fig. 6C). Thus BAY K 8644 significantly shortened the length scale over which nodal lines formed during spatially discordant Cai transient and APD alternans.
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We next investigated whether spatially discordant alternans could create steep enough APD gradients to cause conduction block and initiation of reentry. Under control conditions, as pacing rate increased, 1:1 conduction with normal propagation was followed by 1:1 conduction with lines of block, and finally 2:1 conduction block, in 11 of 37 specimens (29%). Spatially discordant APD or Cai transient alternans preceded the development of lines of conduction block in 81% (9 of 11 specimens), and the site of block occurred near the nodal line in 63% of the cases (7 of 11 specimens). Localized conduction block resulted in initiation of reentry in 6 of 37 specimens (16%).
BAY K 8644 increased the incidence of conduction block during rapid pacing to 79% (22 of 28, P < 0.01), and lines of block were preceded by spatially discordant alternans in all cases. BAY K 8644 also increased the incidence of reentry during rapid pacing from 16 to 57% (16 of 28 specimens, P < 0.05).
The mechanism of conduction block during spatially discordant alternans is illustrated in Fig. 7. After BAY K 8644 exposure, two regions (sites a and b) alternated out of phase in both Cai and APD, separated by a nodal line. Note that the red and green regions of the change (
) in Cai and APD maps (Fig. 7A) match each other, indicating that larger Cai transients are associated with long APD and vice versa. This positive coupling relationship between Cai transient amplitude and APD was observed in all monolayers in which voltage and Cai were simultaneously recorded, both in control and after BAY K 8644. Figure 7B shows that, for beat 3 (black line), APD become shorter as the paced impulse propagated from site a with long APD to site b with short APD. For beat 4 (red line), the APD gradient was reversed. As beat 5 propagated from site a to site b, the gradient in refractoriness left over from beat 4 was too steep for successful propagation, causing a line of conduction block to develop close to the nodal line. The graph of APD along the line ab in Fig. 7B shows that block occurred where the spatial gradient in APD was steepest, reaching 6 ms/mm. Thus 1) spatially discordant alternans can promote a large enough APD gradient over a 22-mm span in monolayers to induce conduction block (18); 2) the nodal line marks the location of steepest spatial APD gradient; 3) conduction block occurs at the nodal line when the new impulse propagates from short-to-long APD direction of the prior beat, which corresponds to the short-to-long Cai transient direction (i.e., positive voltage-Cai coupling) (29); and 4) Cai transient alternans maps correlate highly with APD alternans maps in their ability to predict the site of nodal lines and conduction block.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The novel findings of this study are that spatially discordant APD and Cai alternans 1) can be induced by rapid pacing in a relatively isotropic 2D cardiac tissue in which the entire surface can be optically mapped, so as to exclude unmapped subsurface electrical activity from influencing the outcome, and can create sufficient repolarization gradients to cause conduction block and reentry; 2) share features with intact 3D ventricular muscle, indicating that it originates primarily from dynamic factors such as CV restitution (23, 31) rather than tissue heterogeneity; and 3) are exacerbated by the Ca channel agonist BAY K 8644, as predicted theoretically for an agent that steepens APD restitution, promotes Cai overload, and increases CV restitution.
The following observations strongly support a dynamic, rather than tissue heterogeneity-based, mechanism in this preparation. 1) Under control conditions, CV restitution was more prominent in the 62% of monolayers that transitioned from spatially concordant to spatially discordant alternans, compared with the 38% of monolayers that did not (Fig. 2D). By increasing CV restitution, BAY K 8644 increased the incidence of spatially discordant alternans to 100%. 2) BAY K 8644 also increased the number of nodal lines (i.e., decreased the spacing between nodal lines), consistent with theoretical predictions that the length scale of nodal line spacing is decreased by increasing CV restitution and the amplitude of APD alternans (5, 22). 3) Computer simulations (9, 31) predict that nodal lines generated dynamically by CV restitution should move toward the pacing site as pacing cycle length decreases and should reorient to remain circumferential with respect to the pacing site when the pacing site is changed. In contrast, nodal lines formed as a result of tissue heterogeneity do not share these features (9). In our preparation, we found that nodal lines behaved according to the predictions for the dynamic CV restitution mechanism (Fig. 5), consistent with recent observations on nodal line behavior in intact 3D rabbit ventricular tissue (9). In the case of intact heart tissue, significant macroscopic tissue heterogeneities are unavoidably present. Monolayers, when carefully prepared to be fully confluent, lack gross macroscopic tissue heterogeneity. However, microscopic heterogeneities still exist and may exert important effects. This is evident from our finding that spatially discordant alternans was able to induce localized conduction block (Fig. 8), as required for reentry initiation (rather than circumferential conduction block along the whole wavefront, which cannot induce reentry). The magnitude of the heterogeneity required for this symmetry-breaking effect is unclear. Possibly, it could be very minor at normal heart rates, but amplified by dynamic factors at rapid pacing rates (22), which remains an interesting area to explore in future studies.
Cellular Mechanisms of Repolarization Alternans
Although APD alternans is always dually influenced by both APD restitution steepness (11, 17) and Cai cycling dynamics (2, 3), the onset of APD alternans is driven by whichever factor becomes unstable first (29). In neonatal rat ventricular myocyte monolayers, the following observations favor the Cai-driven instability as responsible for the onset of alternans. First, the onset of alternans occurred at a pacing cycle length at which APD restitution slope was considerably < 1 (Fig. 2), although short-term memory effects (30) or transient alternans (see below) might conceivably account for this. Second, under control conditions, the onset of Ca alternans preceded the detection of APD alternans. This could be due to subtle APD alternans below our detection threshold (0.6–3 ms), but also could be explained if primary Cai transient alternans had balanced effects on Ca-sensitive currents tending to prolong and shorten APD (e.g., Na-Ca exchange and L-type Ca current inactivation, respectively). Although Cai-driven alternans might seem less likely in neonatal rat ventricular myocytes because of their immature SR, recent studies have shown that, after several days in culture (equivalent to the time monolayers were studied here), neonatal ventricular myocytes resemble adult ventricular myocytes more closely than freshly dissociated neonatal myocytes, both histologically and functionally with respect to T tubule and SR Ca cycling properties (10, 32).
The mechanism underlying prominent CV restitution observed in the monolayers is unclear. CV restitution is usually attributed to the kinetics of the Na current's recovery from inactivation. In intact 3D ventricular tissue, CV restitution is typically not engaged until very short DIs, consistent with normally rapid Na current recovery kinetics. In monolayers, however, CV varied almost continuously over a wide range of DIs (Fig. 2D). Although it is possible that cultured neonatal rat ventricular myocytes have much slower Na channel recovery kinetics than adult ventricular cells, another potential explanation may involve heart rate sensitivity of gap junctional conductance, the other key determinant of CV. During rapid pacing, either intracellular acidosis or elevated Cai levels could reduce gap junction conductance and progressively slow CV. The observation that BAY K 8644 exacerbated CV restitution (Fig. 2D) is consistent with this possibility, since BAY K increases diastolic Cai in chick embryonic myocytes (16), and Cabo et al. (1) reported that BAY K 8644 caused CV slowing due to decreased gap junctional conductance in infarcted hearts. A limitation of our study, however, is that we could not reliably measure changes in diastolic Cai in this preparation, as previously noted by Fast et al. (6) in this preparation, although it is possible in other preparations (4, 16).
Spatially Discordant Alternans and Initiation of Reentry
Pastore et al. (18) demonstrated that spatially discordant alternans can produce spatial repolarization gradients steep enough to cause unidirectional conduction block and reentry. We have extended their findings by demonstrating the relevance of the nodal line to the site of conduction block leading to initiation of reentry. Under control conditions, conduction block occurred near the nodal line in 63% of cases. After BAY K 8644, which shortened the spacing between nodal lines, the incidence of conduction block increased and was preceded by spatially discordant alternans in 100% of cases. As predicted theoretically (9, 31) and demonstrated experimentally (Fig. 7A), the nodal line marks the location of steepest APD and Ca transient gradients. The APD gradient at the nodal line in Fig. 7A reached 6 ms/mm, which agrees well with the experimental estimate of >3.2 ms/mm necessary for conduction block measured in intact ventricular tissue (15), as well as with theoretical predictions (21). In monolayers, nodal lines consistently predicted the location of conduction block, such that block occurred when propagation was in the direction of short-to-long APD (i.e., increasing refractoriness) and small-to-large Ca transient of the previous beat. BAY K 8644 increased the incidence of conduction block by shortening the length scale of spatially discordant alternans, which is equivalent to steepening the spatial APD gradient.
Limitations
At any given pacing rate, APD and Cai transient alternans can be either transient or persistent. Any sudden change in heart rate produces transient alternans, which then decays exponentially back to the steady-state APD or Cai transient amplitude for the new heart rate, typically with a beat constant of three to four beats (8). For practical reasons, we used a pacing protocol in which pacing cycle length was decreased every eight beats by 20 ms, which may not unequivocally delineate between transient and persistent alternans. Thus it is possible that the alternans induced by our pacing protocol was really transient alternans maintained by the continually increasing pacing rate. To eliminate very transient alternans, we required that alternans, once started, had to persist throughout the remainder of the pacing protocol (or until conduction block occurred), but we cannot state conclusively that the monolayers exhibited truly persistent alternans. However, from the perspective of arrhythmogenesis, the key factor is whether the gradient in refractoriness caused by spatially discordant APD alternans is steep enough, even transiently, to cause conduction block and initiating reentry. Our findings show unequivocally that the gradients in refractoriness achieved during spatially discordant alternans in 2D monolayers, whether transient or persistent, were sufficient to cause conduction block and reentry, especially after BAY K 8644. Nevertheless, neonatal myocytes have different electrophysiology and Cai cycling features than adult human ventricular myocytes, so that the relevance to the arrhythmogenic consequences of spatially discordant alternans in humans remains speculative. However, if the fundamental dynamics conferred by APD restitution, Cai cycling, and CV restitution play comparable roles in the human heart, they must be considered in the future development of antiarrhythmic therapies.
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GRANTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Present address of M. Valderrábano: Cardiology Division, Methodist DeBakey Heart Center, 6550 Fannin St., Suite 1901, Houston, TX 77030.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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