Am J Physiol Heart Circ Physiol 288: H400-H407, 2005.
First published September 2, 2004; doi:10.1152/ajpheart.00502.2004
0363-6135/05 $8.00
Contrasting effects of ischemia on the kinetics of membrane voltage and intracellular calcium transient underlie electrical alternans
Vikram Lakireddy,
Paramdeep Baweja,
Asma Syed,
Gil Bub,
Mohamed Boutjdir, and
Nabil El-Sherif
New York Harbor Veterans Affairs Healthcare System and Downstate Medical Center, State University of New York, Brooklyn, New York
Submitted 28 May 2004
; accepted in final form 19 August 2004
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ABSTRACT
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Repolarization alternans has been considered a strong marker of electrical instability. The objective of this study was to investigate the hypothesis that ischemia-induced contrasting effects on the kinetics of membrane voltage and intracellular calcium transient (CaiT) can explain the vulnerability of the ischemic heart to repolarization alternans. Ischemia-induced changes in action potential (AP) and CaiT resulting in alternans were investigated in perfused Langendorff guinea pig hearts subjected to 10–15 min of global no-flow ischemia followed by 10–15 min of reperfusion. The heart was stained with 100 µl of rhod-2 AM and 25 µl of RH-237, and AP and CaiT were simultaneously recorded with an optical mapping system of two 16 x 16 photodiode arrays. Ischemia was associated with shortening of AP duration (D) but delayed upstroke, broadening of peak, and slowed decay of CaiT resulting in a significant increase of CaiT-D. The changes in APD were spatially heterogeneous in contrast to a more spatially homogeneous lengthening of CaiT-D. CaiT alternans could be consistently induced with the introduction of a shorter cycle when the upstroke of the AP occurred before complete relaxation of the previous CaiT and generated a reduced CaiT. However, alternans of CaiT was not necessarily associated with alternans of APD, and this was correlated with the degree of spatially heterogeneous shortening of APD. Sites with less shortening of APD developed alternans of both CaiT and APD, whereas sites with greater shortening of APD could develop a similar degree of CaiT alternans but slight or no APD alternans. This resulted in significant spatial dispersion of APD. The study shows that the contrasting effects of ischemia on the duration of AP and CaiT and, in particular, on their spatial distribution explain the vulnerability of ischemic heart to alternans and the increased dispersion of repolarization during alternans.
electrophysiology; arrhythmias; optical mapping
IN RECENT YEARS, repolarization alternans has been considered a strong marker of electrical instability and ventricular tachyarrhythmias (VT) in the clinical setting (17). Alternans of intracellular calcium (Cai) and action potential (AP) has been investigated in both theoretical (3)and experimental models (2, 14) utilizing a variety of techniques. Despite extensive literature on the mechanisms of electrical alternans in normal preparations and in response to marked shortening of cycle length (CL), the pathophysiology of electrical alternans during ischemia has not been well investigated. Ischemia is known to be associated with alterations of AP and Cai kinetics as well as electrical alternans and VT. Previous studies of electrical alternans in experimental models of ischemia utilized analysis of extracellular electrograms (13, 19), monophasic APs (10), or a floating microelectrode technique (5). More recently, recordings of optical signals showing ischemia-induced alternans of either the Cai transient (CaiT) (15) or AP (16) have been reported. However, the correlation between alternans of CaiT and AP during ischemia has not been investigated.
The objective of the present study was to investigate ischemia-induced alterations in AP and CaiT leading to alternans utilizing simultaneous recordings of optical signals of membrane voltage and CaiT. Our results demonstrate that ischemia-induced contrasting changes on the kinetics of membrane voltage and CaiT can explain both the vulnerability of ischemic heart to alternans and the marked spatial heterogeneity of repolarization during alternans.
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METHODS
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This investigation conformed to the current National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, Revised 1996).
Experimental setup.
Guinea pigs (male, 350–450 g, n = 12) were anesthetized with pentobarbital sodium (35 mg/kg), the chest was opened, and heparin (200 U/kg) was injected into the inferior vena cava. The heart was excised, and the aorta was cannulated to allow retrograde perfusion of the coronary vessels with a modified Tyrode solution containing (in mM) 130 NaCl, 25 NaHCO3, 1.2 MgSO4, 4.0 KCl, 20 dextrose, and 1.25 CaCl2, pH 7.4, bubbled with 95% O2-5% CO2. The temperature of the perfusate was maintained at 37.0 ± 0.5°C. The heart was perfused at constant flow rate (12–16 ml/min) with a pump; perfusion pressure was set at 70 mmHg by adjusting the flow of the perfusate.
The heart was placed in a custom-made chamber to reduce movement artifacts, to control the temperature in the surrounding bath, and to hold the stimulating and recording electrodes at selected sites on the heart. The heart was situated against the imaging surface by positioning a Lexan plunger posterior to the imaged plane. The plunger also served to seal the chamber and reduce motion artifacts. A probe was positioned within the chamber to monitor temperature. With this experimental setup, epicardial temperature gradients were <0.2°C (8). The heart was stained with a voltage-sensitive dye (RH237; Molecular Probes), 10–20 µl of a 1 mg/ml solution in DMSO, and was loaded with the Ca2+ indicator rhod-2 AM (molecular Probes), 0–2 mg in 0–2 ml DMSO. Light from two 100-W custom-built, tungsten-halogen lamps was collimated, passed through 520 ± 20-nm interference filters, and focused on the surface of the heart. Fluorescence emitted from the stained heart was collected with a camera lens (85 mm, fl: 1.4 Nikon) and passed through a 45° dichroic mirror with a cutoff of 630 nm (Omega Optical; Brattleboro, VT). Fluorescence images of the heart were then focused on two 16 x 16-element photodiode arrays (C675–103; Hamamatsu, Bridgewater, NJ; 10-M
feedback resistor). The fluorescence image below 630 nm was focused on the "Ca2+ array" after being passed through a 585 ± 20-nm interference filter, and the fluorescence image above 630 nm was refocused on the "voltage array" after being passed through a 715-nm cutoff filter. Each diode had a sensing area of 0.95 x 0.95 mm with a pitch of 1.1 mm (the distance from center to center of neighboring diodes). Each pixel averaged the signals from 
250 cells. The arrays were precisely aligned such that a diode on the voltage array was in exact register with a diode on the Cai array. Outputs from the arrays were amplified, digitized at 2,000 frames/s, and stored in computer memory. Details of data acquisition and analysis have been recently reported (2).
Experimental protocol and data analysis.
The sinus node area was crushed to allow ventricular pacing at relatively long CL. After a stabilization period of 15 min, the heart was paced from the right ventricle (RV) outflow tract at a starting CL of 500–600 ms and then at shorter CLs. The CL was decreased by a 50-ms decrement until a CL of 300 ms, followed by 20-ms decrement until 1:1 capture was lost. The heart was again paced at a CL of 500–600 ms and was subjected to 10–15 min of global no-flow ischemia followed by 10–15 min of reperfusion. At 10 min of ischemia, the CL of stimulation was decreased in 50-ms decrements up to a CL of 300 ms.The effects of a single premature beat or of abrupt shortening of the CL was investigated in four and six experiments, respectively. The changes in the cardiac rhythm, AP, and CaiT were continuously recorded. Activation time at each site was determined from local AP upstroke [(dFv/dt)max]. AP duration (APD) at each site was the interval from (dF/dt)max to the inflection point of the AP downstroke [(d2Fv/dt2)max] (6). During APD alternans at relatively short CL, alternate APs may not repolarize to the baseline level. In this case, the repolarization phase was extrapolated to baseline before an APD was calculated. The duration of CaiT was determined from the maximum first derivative of the CaiT upstroke to 90% recovery of the CaiT to the original baseline. For the purpose of the study, the magnitude of CaiT alternans was measured using an alternans ratio, defined as 1 – B/A, where B is the net amplitude of the shorter CaiT and A is the net amplitude of the larger CaiT (15). Isochronal maps of activation and repolarization and local conduction velocity maps were generated from local activation and repolarization time points of APs as previously described (2). Diodes showing pronounced motion artifacts (an average of 10% per experiment and mainly at the corners of the optical field) were excluded from the analysis.
The term "concordant" CaiT/AP alternans is utilized in this study when the large CaiT is associated with the longer APD.
Statistics.
Results are expressed as means ± SE. Statistical analysis was performed by means of Student's t-test and one-way ANOVA. A value of P < 0.05 was considered significant.
To investigate whether ischemia-induced changes in the APD versus CaiT-duration (D) are spatially homogeneous, a likelihood ratio test of the null hypothesis of homogeneity of variance between APD and CaiT-D scores was constructed by subtracting the –2 x restricted log likelihood statistics resulting from two mixed linear models, both having change scores as a dependent variable and APD and CaiT-D as a fixed factor. In the first model, an unstructured covariance matrix was specified, causing the variance of APD and CaiT-D change scores to be estimated separately, along with their correlation; in the second reduced model, a compound symmetry covariance matrix (i.e., with pooled variances) was specified.
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RESULTS
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CL-dependent alternans in the normally perfused guinea pig heart.
Figure 1A illustrates simultaneous recording of CaiT and AP signals from a representative pixel from the left ventricle (LV) epicardial surface of a Langendorff-perfused guinea pig heart during control recording. The heart was paced from the RV outflow tract at a CL of 450 ms. The upstroke of the CaiT followed the upstroke of the AP with a time delay of 10 ms. The APD measured 220 ms, and the CaiT-D measured 235 ms. Control activation and repolarization isochronal maps are shown in Fig. 1A, bottom. The activation map showed regular isochrones of activation from the RV outflow tract to the LV apex with an apparent conduction velocity of 0.30 m/s. The repolarization map showed that repolarization of AP began at the LV apex and spread toward the LV base and RV outflow tract. These observations are consistent with previous reports (2).
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Fig. 1. Tachycardia-dependent alternans in the normally perfused guinea pig heart. A: simultaneous recordings of intracellular calcium transit (CaiT) and action potential (AP) from a representative pixel during control recording. In this and subsequent figures, the CaiT is colored gray and AP is colored black. The heart was paced from the right ventricle (RV) outflow tract at a cycle length (CL) of 450 ms. The upstroke of the CaiT followed the upstroke of the AP with a time delay of 10 ms. The activation wave proceeded from RV outflow tract to the left ventricle (LV) apex with an apparent conduction velocity of 0.3 m/s. On the other hand, repolarization began at the LV apex and spread toward the LV base and RV outflow tract. B: development of concordant alternans of both CaiT and AP duration (APD) at a CL of 185 ms.
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Figure 1B shows the development of alternans of both CaiT and APD at a CL of 185 ms. During alternans, the small CaiT arose before complete relaxation of the preceding large CaiT, whereas the larger CaiT arose from a lower diastolic level after more complete relaxation of the preceding small CaiT. The CaiT alternans was associated with concordant alternans of APD whereby the larger CaiT was associated with the longer APD (192 ms), whereas the smaller CaiT was associated with the shorter APD (176 ms). The CaiT alternans ratio was 42%. As in the case of CaiT alternans, the upstroke of the short AP arose before complete repolarization of the preceding long AP. On the other hand, the longer AP arose from a more negative diastolic level after complete repolarization of the short AP. In eight control experiments, CaiT alternans was observed at a critical CL of 212 ± 22 ms, and the peak CaiT alternans ratio occurred at a CL of 192 ± 18 ms (Table 1).
Ischemia-induced alterations of AP and CaiT and the effects on CL-dependent alternans.
Ischemia resulted in a varying degree of shortening of APD (from 278 ± 40 ms during control to 206 ± 78 ms after 10 min of ischemia at a Cl of 600 ms, P < 0.01; Table 1). On the other hand, ischemia resulted in a delay of the upstroke of CaiT in relation to the upstroke of the AP (from 8 ± 4 ms during control to 24 ± 11 ms after 10 min of ischemia at a Cl of 600 ms, P < 0.001). It also resulted in broadening of the peak deflection and slowing of the decay of CaiT with an overall increase of the duration of CaiT (from 289 ± 36 ms during control to 368 ± 41 ms after 10 min of ischemia at a Cl of 600 ms, P < 0.001; Table 1). Figure 2 illustrates typical ischemia-induced alterations of AP and CaiT. The ischemia-induced contrasting changes in APD and CaiT-D could result in a situation in which full repolarization of AP could occur at a time when the decay of CaiT has just started. The introduction of a pacing stimulus at a relatively short CL could succeed in generating a full AP at a time before the decay of the preceding CaiT and would thus result in a markedly reduced CaiT. This would perturb the classic negative feedback system of normal Cai autoregulation (7) and result in alternans of subsequent CaiT amplitude. This process could follow a gradual or abrupt increase of the heart rate (Fig. 2). A similar mechanism could occur after the introduction of a single premature beat that results in shortening of the CL by 25–50% compared with the basic CL not associated with alternans. This is illustrated in Fig. 3, where alternans developed during stimulation at a basic CL of 600 ms after the introduction of a single premature beat at a CL of 300 ms.
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Fig. 2. Typical ischemia-induced alterations of AP and CaiT and their relationship to electrical alternans. A: control recordings from a representative pixel. The heart was paced at a CL of 600 ms. B: recordings obtained after 10 min of no-flow ischemia. The first 3 beats were at a CL of 600 ms. Ischemia resulted in significant shortening of APD from 330 ms during control to 160 ms with no significant change in AP amplitude. On the other hand, ischemia resulted in delay of the upstroke of CaiT in relation to the upstroke of the AP from 8 ms during control to 36 ms. Furthermore, ischemia resulted in broadening of the peak deflection and slowing of the decay of CaiT with an overall increase of the duration of the CaiT from 345 ms during control to 436 ms after ischemia. Faster pacing was abruptly introduced at CL of 300 ms (marked by arrow). The introduction of a pacing stimulus at the shorter CL of 300 ms succeeded in capturing the heart and generating an AP. However, the AP upstroke coincided with a time before the decay of the preceding CaiT and generated a markedly reduced CaiT and the onset of alternans of subsequent CaiT amplitude (alternans ratio of 50%). The CaiT alternans was associated with concordant alternans of APD whereby the larger CaiT was associated with the longer APD (280 ms), whereas the smaller CaiT was associated with the shorter APD (150 ms). When the slower pacing cycle was resumed (C), the CaiT immediately returned to baseline amplitude and duration. Time scale is the same for A–C.
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Fig. 3. Recordings from a different experiment showing that a single premature stimulus could perturb the normal CaiT autoregulation and initiate CaiT alternans at the same CL that was not associated with alternans. A: control recording where the heart was paced at a CL of 600 ms. B: recording obtained after 10 min of global ischemia. The first 2 beats at a CL of 600 ms show the typical alterations in APD and CaiT described in Fig. 2. Ischemia was associated with a shortening of APD from 309 ms during control to 248 ms, whereas it resulted in delay of the upstroke and slowing of the decay of CaiT with an increase of the duration from 320 ms during control to 380 ms. A single stimulated beat at CL of 300 ms resulted in an AP that was associated with a markedly reduced CaiT. After the premature beat, the heart continued to be stimulated at a CL of 600 ms, which was then associated with CaiT alternans (alternans ratio of 32%). However, in contrast to Fig. 2, CaiT alternans was associated with minimal or no discernible alternans of APD. Time scale is the same for A and B.
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Spatial heterogeneity of APD and electrical alternans.
Table 1 shows that the CLs at both the onset and peak CaiT alternans was significantly longer during 10 min of ischemia compared with control. Table 1 also shows ischemia-induced alterations in APD and CaiT-D. Ischemia resulted in marked heterogeneity of the shortening of APD at different sites in the epicardial optical field. On the other hand, there was less spatial variation in the degree of prolongation of CaiT-D at various sites. This is illustrated in Figs. 4 and 5. Figure 4 shows recordings from two separate pixels from the same experiment. Ischemia resulted in a similar degree of lengthening of CaiT-D but in markedly different degrees of shortening of APD. A critical short CL resulted in the same degree of CaiT alternans at the two sites. However, APD alternans did not develop at the site with the short APD, whereas significant APD alternans occurred at the site with the long APD. Figure 5 shows repolarization maps of two consecutive beats at a pacing CL of 300 ms from the same experiment shown in Fig. 4. Figure 5 illustrates the marked increase in spatial dispersion of repolarization of alternate beats. In the 10 experiments in which APD alternans developed, the dispersion of APD in the epicardial optical field in the absence of alternans was 67 ± 38 ms (n = 4) at CL of 500 ms and changed to 68 ± 28 and 148 ± 52 ms (n = 6) in alternate beats at CLs of 300–400 ms during alternans. The difference in the dispersion of APD in the absence of alternans and during the alternate beat with the large dispersion of APD during alternans was highly significant (P < 0.001).
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Fig. 4. Recording from a different experiment to illustrate the effects of spatial heterogeneity of ischemia-induced shortening of APD on the development of electrical alternans and dispersion of repolarization. A and B: sequential recordings from two separate pixels in the epicardial optical field. Control recordings during pacing at CL of 600 ms are shown on top and demonstrate that the AP and CaiT at both sites have largely similar configuration and duration. The recordings on middle top were obtained after 10 min of no-flow ischemia while pacing was maintained at CL of 600 ms. Pixel A showed a shortening of APD from 305 ms during control to 230–250 ms in alternate beats during ischemia. On the other hand, pixel B showed marked shortening of APD from 310 ms during control to 150 ms during ischemia. The increase in CaiT-D at both sites was comparable. CaiT alternans developed at both sites with an alternans ratio of 25% at site A and 16% at site B. CaiT alternans was associated with 20 ms alternans of APD at site A but no discernible alternans at site B. The overall dispersion of APD in the optical field was 80 and 100 ms in alternate beats. The recordings on middle bottom were obtained 45 s later when the pacing CL was decreased from 600 to 300 ms. Marked CaiT alternans of approximately equal degree developed at both pixels (alternans ratio of 55% at site A and 58% at site B). However, the CaiT alternans was associated with marked concordant APD alternans at site A but no discernible alternans at site B. The overall dispersion of APD in the optical field was 68 and 176 ms in alternate beats. The recordings on bottom were obtained after 2 min of reperfusion while pacing was maintained at a CL of 300 ms and show complete resolution of CaiT and APD alternans.
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Fig. 5. Repolarization maps of two consecutive beats at a pacing CL of 300 ms from the same experiment shown in Fig. 4. AP recordings from pixels A and B as well as from a third pixel, pixel C, are shown on the right. The 3 pixels represent recordings from the base, center, and apex of the LV. The AP recordings from pixels A and B are superimposed on the right bottom and show that the dispersion of APD in alternate beats varied from 45 to 162 ms. The repolarization maps of two subsequent beats drawn at 20-ms isochrones are shown on the left. The left top map shows marked spatial dispersion of APD as reflected in the crowded isochrones. The APD was relatively short at the center of the lower half of the optical field and was much longer at basal sections and both right and left apical sections resulting in at least 3 separate zones of marked gradient of repolarization. The left bottom map of the subsequent beat shows a more organized repolarization pattern and a much less degree of spatial dispersion of repolarization.
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Figure 6 illustrates the time dependence of spatial dispersion of both APD and CaiT-D after ischemia at a CL of 600 ms from the same experiment shown in Figs. 4 and 5. During control, the spatial distribution patterns of APD and CaiT-D were largely similar. Ischemia resulted in spatial heterogeneity of the shortening of APD that gradually increased during 10 min of ischemia. On the other hand, ischemia resulted in a monotonous, gradual spatially homogeneous lengthening of CaiT-D. In five experiments, the variances of the change scores of APD versus CaiT-D became significant after 7 min of ischemia and increased further after 10 min of ischemia (
2 = 90.3, df = 1, P < 0.001). On the other hand, there was no significant correlation between the change scores (r = –0.21, P = 0.168).
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Fig. 6. Recordings obtained from the same experiment shown in Figs. 4 and 5 that illustrate the time dependence of spatial dispersion of both APD and CaiT-D after ischemia at a CL of 600 ms. During control, the spatial distribution patterns of APD and CaiT-D were largely similar. Ischemia resulted in spatial heterogeneity of the shortening of APD that increased gradually during 10 min of ischemia. On the other hand, ischemia resulted in a monotonous gradual spatially homogeneous lengthening of CaiT-D.
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CL-dependent electrical alternans after ischemia was not associated with conduction alternans. This is illustrated in Fig. 7, obtained from the same experiment shown in Figs. 4–6, and shows the activation maps of two consecutive beats with marked alteration of APD.
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Fig. 7. Activation maps of 2 consecutive beats from the same experiment shown in Figs. 4 and 5 that illustrate the absence of conduction alternans. This is demonstrated by the superimposition of the upstroke of AP of alternate beats associated with APD alternans and a similar activation pattern of the two beats. The activation pattern after ischemia (B) was remarkably similar to control (A) but with a moderate decrease of conduction velocity.
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APD/CaiT alternans and arrhythmogenesis.
APD/CaiT alternans developed in 10 of 12 experiments. On the other hand, one or more episodes of VT developed in 8 of 12 experiments during ischemia or reperfusion. However, only in three experiments did the arrhythmia develop during manifest epicardial alternans. It was not uncommon during reperfusion to observe amelioration or disappearance of "epicardial" alternans to be followed later by the development of VT initiated by a premature beat.
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DISCUSSION
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The present study showed that ischemia results in contrasting changes in the duration of the AP and CaiT. The rise in extracellular potassium (Ko) secondary to opening of sarcolemmal ATP-sensitive potassium channels results, among other factors, in a reduction of membrane resting potential and shortening of APD (5). In contrast, ischemia results in a delay of the upstroke, broadening of the peak, and delay in the decay of the CaiT. This creates a situation in which full repolarization of AP occurs at a time when cytosolic Cai from the preceding transient is still elevated. This makes the ischemic heart particularly vulnerable to the development of CaiT alternans after a single "shorter cycle" that can result in a full AP but a reduced CaiT. The increased sarcoplasmic reticulum (SR) Ca content and lower cytosolic Ca are then expected to result in a large CaiT for the next beat. This perturbs the normal negative feedback Cai autoregulation and results in CaiT alternans. The same mechanism applies in response to a series of shorter CLs rather than a single shorter CL. CaiT alternans during ischemia is CL dependent. However, in contrast to fast pacing-induced CaiT alternans in the normal guinea pig heart, during ischemia CaiT alternans occurs at relatively long CLs. This is to be expected given ischemia-induced depression of Cai kinetics. The activity of both the SR Ca-ATPase and the ryanodine receptor (RyR) are regulated by ATP. During ischemia, therefore, when ATP falls, uptake of Ca into the SR as well as the release would be compromised (7). It is well established that the open probability of the RyR can be effected by metabolic inhibition, which was shown to delay the upstroke and prolong the duration of the CaiT (4). Of potential relevance to ischemia, the open probability is decreased by acidification or a decrease of cytoplasmic ATP concentration (7).
Relationship between CaiT alternans and APD alternans.
Theoretical and experimental observations suggest that, at least, three mechanisms can result in APD alternans. One mechanism involves the physiological APD restitution whereby the APD is dependent, among other factors, on the preceding diastolic interval (9). A second mechanism implicates conduction velocity restitution as a possible mechanism for APD alternans and especially for the transition from concordant to discordant alternans (14). The third mechanism proposes that CaiT amplitude controls the duration of the AP through effects on one or several Ca-regulated ionic currents. A rise in cytosolic Ca is known to produce inward current through the Na/Ca exchanger (12) as well as outward current carried by Ca-activated chloride current (20) and faster inactivation of the L-type Ca current through Ca-mediated inactivation (11). Consequently, CaiT alternans could, at least theoretically, explain either concordant APD alternans, in which the taller CaiT is associated with the longer APD or discordant alternans in which the reverse is true (16).
Our observations of the effects of ischemia on cardiac activation and repolarization patterns confirm previous reports of the effects of ischemia (1) or hypoxia (18). Similarly, our observations on the effect of ischemia on the CaiT have been previously reported (7, 15). Of particular interest in the present study, however, is the difference in the spatial distribution of these changes in the intact heart, which could only be observed when simultaneous recordings of AP and CaiT were obtained. The spatially heterogeneous shortening of APD during ischemia coupled with a relatively more spatially homogeneous lengthening of CaiT-D was identified as playing a major role in the development of APD alternans and the greater degree of dispersion of APD during ischemia. The mechanism of the spatially heterogeneous shortening of APD during ischemia is related, among other factors, to the degree of the local increase of Ko, which is a net outcome of intracellular potassium efflux and washout from the extracellular space (11). On the other hand, ischemia-induced lengthening of duration of CaiT is related primarily to depressed kinetics of Cai cycling, which, as shown in the present study, seems to be more spatially homogeneous. The study thus deemphasizes, but does not exclude, the role of alternation of the magnitude of CaiT in modulating ADP and ADP alternans in the setting of acute ischemia.
Alternans and arrhythmogenesis during ischemia.
Alternans of both CaiT and APD is a common occurrence after ischemia (10 of 12 experiments). The same is true for VT (8 of 12 experiments). However, VT was observed to occur in the presence of "epicardial" APD alternans in only three of eight experiments. It is of course possible that APD alternans was present in sites outside the epicardial optical window. It is, however, more likely that increased dispersion of repolarization associated with APD alternans is only one of the substrates for reentrant VT during ischemia, besides impaired conduction, decreased excitability, and reduced cellular coupling. These factors were not investigated in the present study.
Study limitations.
The algorithms for measurement of APD and CaiT-D provide reasonably accurate data during control (6). After ischemia, at relatively short CL, alternate APs may not repolarize to baseline. In this case, the repolarization phase was extrapolated to baseline before APD was calculated. Furthermore, ischemia resulted in slowing of the upstroke of the CaiT, which made determination of the first derivative of the upstroke more difficult. However, the difficulties in precise estimation of APD and CaiT-D after ischemia do not detract from the main conclusions in the present study, i.e., ischemia induces spatially heterogeneous shortening of APD versus spatially homogenous lengthening of CaiT-D.
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GRANTS
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This study was supported in part by Veterans Affairs Cardiovascular Research Enhancement Award Program (to N. El-Sherif).
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FOOTNOTES
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Address for reprint requests and other correspondence: N. El-Sherif, Downstate Medical Center, State Univ. of New York, 450 Clarkson Ave., Box 1199, Brooklyn, NY 11203 (E-mail: nelsherif{at}aol.com)
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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