Long-term cardiac memory in canine heart is associated with the evolution of a transmural repolarization gradient

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1 Cardiovascular Research 74 (2007) Long-term cardiac memory in canine heart is associated with the evolution of a transmural repolarization gradient Ruben Coronel a,, Tobias Opthof a,b, Alexei N. Plotnikov d, Francien J.G. Wilms-Schopman c, Iryna N. Shlapakova d, Peter Danilo Jr. d, Eugene A. Sosunov d, Evgeny P. Anyukhovsky d, Michiel J. Janse a, Michael R. Rosen d a Experimental and Molecular Cardiology Groups, Academic Medical Center, University of Amsterdam, The Netherlands b Department of Medical Physiology, University of Utrecht, The Netherlands c Interuniversity Cardiology Institute of the Netherlands, Utrecht, The Netherlands d Center for Molecular Therapeutics, Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, U.S.A. Abstract Received 5 October 2006; received in revised form 20 February 2007; accepted 22 February 2007 Available online 28 February 2007 Time for primary review 25 days Objective: The contribution of regional electrophysiologic heterogeneity to the T-wave changes of long-term cardiac memory (CM) is not known. We mapped activation and repolarization in dogs after induction of CM and in sham animals. Methods and results: CM was induced by three weeks of AV-sequential pacing at the anterior free wall of the left ventricle (LV), midway between apex and base in 5 dogs. In 4 sham controls a pacemaker was implanted but ventricular pacing was not performed. At 3 weeks, unipolar electrograms were recorded (98 epicardial, 120 intramural and endocardial electrodes) during atrial stimulation (cycle length 450 ms). Activation times (AT) and repolarization times (RT) were measured and activation recovery intervals (ARIs) calculated. CM was associated with 1) deeper T waves on ECG, with no change in QT interval; 2) longer activation time at the site of stimulation in CM (29.7±1.0, X±SEM) than sham (23.9±1.3 ms pb0.01); 3) an LV transmural gradient in repolarization time such that repolarization at the epicardium terminated 12.4±2.4 ms later than at the endocardium pb0.01), in contrast to no gradient in shams (2.7±4.2 ms); in memory dogs, the repolarization time gradient was greatest at sites around the pacing electrode varying from 13.1±2.3 ms to 25.5±3.8 ms; 4) more negative left ventricular potentials at the peak of the body surface T wave ( 4.9±0.8 vs 2.2±0.4 mv; pb0.05) but no altered right ventricular epicardial T-wave potentials. ARIs did not differ between groups. Right ventricular activation was delayed but was not associated with altered repolarization because of compensatory shortening of the right ventricular ARIs. Conclusion: CM-induced T-wave changes are caused by evolution of transmural repolarization gradients manifested during atrial stimulation that are maximal near the site of ventricular pacing European Society of Cardiology. Published by Elsevier B.V. All rights reserved. Keywords: Long-term cardiac memory; Mapping; Activation; Repolarization; T wave; Heterogeneity 1. Introduction Corresponding author. Department of Experimental Cardiology, Experimental and Molecular Cardiology Groups, Academic Medical Center,M0-107, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Tel.: ; fax: address: r.coronel@amc.uva.nl (R. Coronel). Cardiac memory (CM) is characterized by an altered T- wave morphology induced by ventricular pacing that persists after resumption of sinus rhythm, or atrial pacing [1 4]. The T-wave vector on return to sinus rhythm or atrial pacing tracks the QRS vector during ventricular pacing [4]. A /$ - see front matter 2007 European Society of Cardiology. Published by Elsevier B.V. All rights reserved. doi: /j.cardiores

2 R. Coronel et al. / Cardiovascular Research 74 (2007) Fig. 1. Example of a body surface ECG (top, lead II) and two local unipolar electrograms, one recorded from the epicardium of the right ventricle (RV, middle), the other from the epicardium of the left ventricle (LV, bottom). The beginning of the Q-wave on ECG (dotted line marked ref ) served as a reference time for activation time (AT) and repolarization time (RT) measurements. AT was the interval between the reference time to the maximum negative dv/dt in the local electrogram, RT the interval between the reference time and the maximum positive slope in the electrogram T wave (both for positive and negative T waves, RV and LV, respectively [16]). Activation Recovery Intervals (ARIs) are the difference between the RT and AT. distinction is made between short-term memory (which we have standardized using 1 2 h of ventricular pacing) [3,5] and long-term memory (weeks of ventricular pacing) [4]. Recent studies have identified different mechanisms for short- and long-term memory: the former appears to result from altered ion channel trafficking [6], whereas the latter is preceded and accompanied by altered ion channel and gap junctional gene transcription and protein levels [7 9]. We have previously reported that the T-wave changes characteristic of short-term memory are associated with an altered apicobasal repolarization gradient, but transmural gradients are not seen in this case [10]. However, microelectrode studies in epicardial cells isolated after prolonged ventricular pacing demonstrated delayed repolarization [4] and this suggests that a transmural gradient may evolve in long-term memory. Based on the above, we hypothesize that long-term memory is associated with evolution of a transmural repolarization gradient resulting from an epicardial repolarization delay. We hypothesize further that this might result from delayed epicardial activation [9] and that activation and repolarization changes would be expressed regionally rather than globally [11]. We therefore designed experiments to define changes in activation times, activation recovery intervals and repolarization times in sham-operated dogs (pacemaker and electrodes implanted but ventricles not

3 418 R. Coronel et al. / Cardiovascular Research 74 (2007) Fig. 2. Lead I of ECG and frontal plane VCG during atrial (left panel) and ventricular pacing (middle panel) at 130 beats/min of a dog at days 0 and during atrial pacing after 21 days of ventricular pacing (day 21). Note that on day 21 there are 1) a deeper T wave on ECG reflecting the negative QRS complex during ventricular pacing, 2) rotation of the average T-wave vector in the direction of the paced QRS, 3) an increase in the average T vector amplitude, and 4) displacement of the average T vector from its control position. Calibrations on ECG=1 mv and 25 mm/s; VCG crosshairs are 0.5 mv. paced) and dogs paced from the ventricles for 3 weeks to induce long-term memory. We found that epicardial activation is delayed in regionally dispersed fashion in the setting of memory and is associated with delayed repolarization times, creating a transmural gradient that is not present in the sham animals. Fig. 3. Example of the three-dimensional distribution of transmural and epicardial activation times (AT) and repolarization times (RT) in the hearts of one sham dog and one memory dog during atrial pacing. Transmural AT and RTs are depicted at three levels: basal, middle (mid) and apical. RV: right ventricle; LV left ventricle. Lines are isochrones. Colors indicate isochronal classes (scale in ms shown below). Site of stimulus electrode is indicated by stimulus sign. Note that LV RTs are delayed in memory relative to sham, and that transmural LV RT gradients are larger.

4 R. Coronel et al. / Cardiovascular Research 74 (2007) Methods The experimental protocol complied with the Guide for the Care and use of Laboratory Animals published by the US National Institutes of Health (NIH publication 85-23, revised 1996) and was approved by the Columbia University Animal Care and Use Committee. The protocol for inducing longterm memory has been described before [5]. In brief, 9 mongrel dogs of either sex weighing 22 to 26 kg were anesthetized with propofol 6 mg/kg and inhalational isoflurane (1.5% to 2.5%). After intubation and ventilation and under sterile conditions, a thoracotomy was performed at Fig. 4. Pooled epicardial ATs and RTs from all experiments during atrial pacing. The epicardial surface of each heart was divided into 9 LV and 8 RV segments: anterior basal, middle and apical; lateral basal, middle and apical and posterior basal, middle and apical. The position of the stimulus electrode is indicated by the stimulus sign. Asterisks indicate statistically significant difference between sham and memory.

5 420 R. Coronel et al. / Cardiovascular Research 74 (2007) the fifth left intercostal space. Epicardial unipolar leads (model 4965, Medtronic) were attached to the basal-lateral left ventricular wall and left atrial appendage. The leads were connected to a dual-chamber pacemaker (Prodigy DR, Medtronic) affixed subcutaneously. One platinum bipolar electrode was sewn to the left atrial appendage to permit atrial pacing. Fourteen-21 days of recovery were allowed post-surgery to permit ECGs and vectorcardiograms to stabilize before starting the pacing protocol. The 5 memory animals then underwent atrioventricular sequential pacing (DDD mode) with a minimal atrial rate of 120 bpm for 21 days (PR interval 50 to 60 ms to minimize competing and fusion beats); the 4 sham animals received pacing from the atrium (AAI mode), with a minimum rate of 120 bpm. In the dogs paced from the ventricles, less than 2% of beats originated from intervening atrial beats. Lower and upper tracking rates were 120 and 150 beats/min. Six standard lead ECGs were recorded from the extremities prior to and after surgery and twice weekly during the pacing protocol and in the sham animals as well. Cardiac memory was quantified vectorcardiographically as changes in T-wave vector angle, amplitude and displacement, as previously described by us [4,5]. After 3 weeks, the animals were anesthetized, intubated and ventilated as above for the performance of terminal experiments. The left femoral artery was catheterized to monitor blood pressure. A heating pad was used to maintain body temperature. The heart was suspended in a pericardial cradle via a thoracotomy at the fifth left intercostal space. Temperature was monitored at the anterior epicardial surface and deep in the pericardial cradle. The maximum difference in temperature at these 2 sites, as well as at the maximum variation at the same site during the course of any one experiment was less than 0.5 C. Epicardial sock electrodes (98 electrode terminals organized in 7 strips each, harboring 2 rows of 7 electrodes separated by 1.5 cm between rows and columns) were sutured onto the surface of right and left ventricles. Depending on the cardiac anatomy the strips were positioned at variable distances (1 2 cm at the base and touching at the apex). In addition, 24 needle electrodes (0.5 mm diameter) with terminals 1, 5, 9 and 13 mm deep to the epicardial surface were inserted into the left ventricular wall, and 12 needle electrodes with terminals at 1 and 5 mm deep to the epicardial surface were inserted in the right ventricular wall (at 5 mm distance from the epicardium 12% of the RV electrodes recorded cavity potentials). This yielded a maximum of 120 recordings of unipolar electrograms per experiment. Because the deepest electrode terminal in the left ventricle sometimes recorded a cavity potential, the actual number of electrograms was usually less than 120. The electrograms, together with 2 surface ECGs (leads I and II) were simultaneously recorded using a personal computerbased data acquisition system as described previously [11]. The reference signal was derived from a ground electrode connected to the mediastinum. Recordings were made during atrial pacing at CL=450 ms in sham and memory dogs (in the latter with the ventricular pacemaker switched off). The signals were filtered (DC-1 khz (3dB)) and selected 2 s episodes were stored on the hard disk of the computer. Sampling interval was 0.5 ms. Analysis of the signals was done off-line with a custom made data analysis program [12]. Activation times (AT) were measured as the interval between the onset of deviation of the isoelectric line preceding the body surface QRS complex and the time of maximum negative slope of the local unipolar QRS complex. Repolarization times (RTs) were measured as the interval between the beginning of the body surface QRS complex and the maximum positive slope of the local electrogram T wave [13 16]. Activation recovery intervals (ARI) were calculated as the difference between AT and RT (Fig. 1). Fig. 5. Activation times (ATs) of the left anterior middle segment in the sham group (unfilled circles) and the memory group (filled circles) during atrial pacing. Dots indicate data from individual hearts. Activation is from endo- to epicardium. Activation delay is greater in the memory dogs than in the sham dogs, but is significantly greater only at the subepicardium (0 mm).

6 R. Coronel et al. / Cardiovascular Research 74 (2007) Fig. 6. Left ventricular transmural gradients in repolarization time (RT) depicted at three levels: basal, middle (mid) and apical and at the left anterior (LA), left lateral (LL) and left posterior (LP) sites. In none of the segments during sham experiments were the RT gradients statistically different from 0. In contrast RT gradients did differ significantly from 0 in 4 segments in the memory group (#). Moreover, the gradient was significantly larger in the memory dogs than in the sham animals in two segments (asterisks). Scale is at the right in ms. Epicardial isopotential maps were constructed at the moment of the peak of the T wave in the surface electrocardiogram Statistical analysis Although we made up to 218 measurements per dog, electrograms could not be recorded from every available electrode position in each heart, for technical reasons. To correct for missing values, we averaged the available measurements in the left ventricular wall over 9 segments ((left anterior base, middle, and apex, left lateral base, middle and apex, left posterior base, middle and apex) and 5 depths (epicardial, 1 mm, 5 mm, 9 mm, and 13 mm, thus yielding a total of 45 regional sites in the left ventricle. The data from two closely appositioned left anterior electrode strips were combined in the left anterior segments. Data were similarly assigned to sites in the right ventricle. One way ANOVA was the principal statistical method for exploring differences between sham and memory dogs. The paired t- test was used for comparison of QT-intervals within the same animal at the start and end of the experiment. Results are presented as means±sem. p b0.05 was considered significant. 3. Results Fig. 2 shows a lead I electrocardiogram during atrial and ventricular pacing at 130 beats/min of a dog at day 0, and during atrial pacing after 21 days of ventricular pacing to induce memory. Prominent changes in the T wave and its vector on day 21 are noted that follow the average vector direction and amplitude of the paced QRS complex. There were no significant changes in the QT interval, either in the four sham-operated dogs (day 0=222±7 ms, 3 weeks=218± 5 ms) or the five memory dogs (day 0=228±8 ms, 3 weeks=231±14 ms, all NS). The quantification of the vectorcardiogram revealed a T vector displacement at 3 weeks in the sham animals of 0.15±0.05 mv; this was altered from that in the memory animals (0.50±0.09 mv, p b 0.05), as we have demonstrated previously [4,5]. Fig. 3 depicts the three-dimensional distribution of transmural and epicardial ATs and RTs in the heart of one sham dog and in the heart of one memory dog. In both hearts activation proceeds from endocardium to epicardium. Epicardial activation occurs earliest in the right ventricle and latest at several sites in the left ventricle, as has been demonstrated earlier [17]. In the memory heart it is later than in the sham heart and results from slower transmural activation. The repolarization patterns of the hearts show large differences between right and left ventricles. These result from longer ARIs in the left than in the right ventricular wall. Although differences in right ventricular repolarization between the sham and memory heart are not apparent, left ventricular epicardial repolarization appears to be delayed in the memory heart, reflecting left ventricular activation delay in the latter. In the transmural sections of the memory heart a larger transmural repolarization gradient than in the sham heart is observed (see crowding of isochrones). Fig. 4 shows the pooled epicardial data from all hearts. Data were pooled in 9 left ventricular and 8 right ventricular segments (on the right anterior apical segment electrode strips were overlying those from the left anterior apical segment). In both sham and memory hearts activation was early in the right and late in the left ventricular epicardium (cf also Fig. 3 and [17]). In comparison with the sham hearts, activation is delayed inhomogeneously in the memory hearts. The AT differences reached statistical significance in 3 right and 1 left ventricular segments (asterisks). The

7 422 R. Coronel et al. / Cardiovascular Research 74 (2007) latter (the left anterior middle segment) is adjacent to the approximate location of the ventricular pacing electrode across experiments, as shown in Fig. 4. These events did not lead to delayed right ventricular repolarization. However, in the left ventricle RTs were significantly prolonged in two segments (left lateral basal and middle segments) adjacent to the stimulus electrode. At the apex the difference between left and right ventricular positions can be problematic (see also Fig. 3). The significant but small difference in RT observed in the right lateral apical segment may be explained by this. No significant differences in ARIs were observed (LV: 201.7±2.6 vs 199.4±2.2 ms; RV: 179.8±2.7 vs 168.8± 4.9 ms (sham and memory, respectively)). The averaged transmural activation times in the left anterior middle segment are shown for each individual heart in Fig. 5. As expected, activation progresses from endocardium to epicardium. The subepicardium (0 mm) of all memory hearts was activated later than the sham hearts ( p b 0.05). In other transmural layers overlap occurred between memory and sham hearts. Because repolarization times were only longer at the epicardium of the left ventricular segments, and not in the right ventricle, transmural gradients were assessed in the 9 left ventricular segments, as shown in Fig. 6. In sham hearts transmural gradients in repolarization times were not significantly different from zero in any segment and the averaged (all 9 segments) difference between epicardial and endocardial RT was 2.7 ±4.2 ms. In memory hearts the averaged difference in all segments was 12.4±2.4 ms ( pb0.01 vs sham). In 5 segments the transmural gradient was even larger, varying from 13.1±2.3 to 25.5±3.8 ms (Fig. 6). In four of these segments the gradient was significantly different from zero, and in two segments (left lateral apex and left posterior apex) also differed significantly from sham hearts. Fig. 7A shows isopotential maps of the epicardial surfaces of right and left ventricles, at the moment of the peak of the T wave in the body surface electrocardiogram (lead II). There are no evident differences between the sham dog and the memory dog on the right ventricular wall. The left ventricular epicardial potentials are far more negative in the memory dog. Fig. 7B shows the averaged right and left ventricular epicardial potentials, recorded at the peak of the T wave in the body surface ECG, for all sham and memory dogs. On the right ventricle there are no differences, but on the left ventricle the difference is significant ( p b 0.025). Fig. 7. Left panels (A): Isopotential maps from the epicardial surface of right (RV) and left ventricle (LV) at the moment of the peak of the T wave in the body surface ECG (lead II) of a sham dog (upper panel) and a memory dog (lower panel). Note the deeper negative potentials in the left ventricle of the memory dog. Lines are isopotential lines (2 mv). Colors represent isopotential classes (scale shown in the lower panel). Right panel (B): Averaged epicardial potentials at the moment of the peak of the T waves in the ECGs of the 4 sham dogs and the 5 memory dogs. There is no difference between the potentials from the right ventricle (RV) of sham and memory dogs, but the difference in left ventricular (LV) potentials is significant (pb0.05). The local electrograms below each map are recorded from the sites marked A, B, C, and D.

8 R. Coronel et al. / Cardiovascular Research 74 (2007) Discussion The major findings of this study are: long-term memory is associated with 1) deeper T waves in the body surface ECG; 2) a prolongation of activation time; 3) a transmural gradient in repolarization time in the left ventricle with repolarization at the epicardium being later than at the endocardium; and 4) more negative left ventricular epicardial potentials at the moment of the peak of the T wave in lead II of the body surface ECG. The activation delay is an important factor in determining the endocardial to epicardial gradient in repolarization time in the left ventricle. The early activation of the subepicardium of the right ventricular free wall (in both sham and memory dogs) in comparison with the left ventricular subepicardium can be attributed to the lesser right ventricular wall thickness. We attribute the increased left ventricular epicardial delay in memory dogs to the remodelling of gap junctions caused by altered ventricular activation during ventricular pacing. Both a decrease in epicardial connexin43 protein and lateralization of epicardial connexin43 staining were previously noted close to the pacing site in dogs paced into long-term memory [9]. These changes diminished with distance from the pacing site. Because we have not measured regional Cx43 distribution in these experiments, we cannot correlate a decrease Cx43 with altered activation in a regional manner, but the results are consistent with our earlier study [9]. In short-term memory the QT interval shortened by about 10 ms, ARIs shortened globally by about 10 ms as well, activation times did not change, and the slope of phase 3 of the monophasic action potential increased [10]. In contrast, long-term memory did not cause shortening of the QT interval in the ECG and did not cause global changes in ARIs in the left ventricle. Whereas in short-term memory the changes occur throughout the left ventricle [10] in the present study there were regional differences. This is in agreement with earlier studies, in which the largest changes were found at sites close to the pacing electrode [7,11]. The observation of regional changes of repolarization times related to the site of pacing suggests this is associated with/ induced by the locally altered activation pattern during ventricular pacing. When the pacing site is altered action potential duration lengthens near the pacing site and shortens at far sites. This may in itself explain at least in part the changes in repolarization. However, we did not find significant changes in ARIs during long-term memory. It is conceivable that ventricular pacing has largely localized effects because as soon as the Purkinje system is activated a more or less normal activation sequence from endo- to epicardium occurs. We speculate that repolarization changes induced by long-term memory occur at sites located preferentially in the dominant epicardial fiber direction extending from the pacing site [18]. In short-term memory the deeper T waves in the ECG were ascribed to the increased slope of phase 3 of the action Fig. 8. A. Hypothetical and schematic action potentials at the endocardium (endo, solid lines) and epicardium (epi, dotted lines) in right and left ventricle (RVand LV) of sham (blue) and memory dogs (green). Intracellular potential gradients between endocardium and epicardium at the moment indicated by the red vertical line are schematically depicted (boxes in each panel). Numbers denote transmembrane potential in mv. In the right ventricle the gradients do not differ between sham and memory dogs, and the T waves in the local electrograms are the same in sham and memory dogs (panel B). Because of the development of a transmural gradient in the left ventricle after induction of long-term memory, the potential gradient at the moment of the vertical line is much larger compared to sham dogs. This accounts for the large potential sink in the epicardial extracellular space of the memory dog and the deeper negative T wave in the epicardial electrogram. To facilitate the visualization of the potential gradients during phase 3 of the action potential, the RT differences in the LV sham and memory hearts were slightly exaggerated.

9 424 R. Coronel et al. / Cardiovascular Research 74 (2007) potential [10]. In long-term memory the explanation is different. This is illustrated in Fig. 8: hypothetical action potentials are drawn from endocardium and epicardium at right and left ventricles (panel A), in sham dogs and memory dogs. Intracellular potential gradients between endocardium and epicardium at the moment of the red dotted vertical line are schematically depicted, as well as local electrograms at the epicardium (panel B). Despite the activation delays in the right ventricles of the memory dogs, repolarization times are not altered because of the compensatory slight shortening of ARIs. Consequently, the potential gradients are similar in sham and memory dogs, and the local T waves in the epicardial electrograms are the same. In contrast, because of the development of an endocardial to epicardial repolarization gradient in the left ventricles of the memory dogs, the intracellular potential gradient at the moment of the vertical line is much larger compared to sham dogs. Therefore, the local sink in the epicardial extracellular space is much larger, accounting for the deeper negative T wave in the local electrogram, as well as in the body surface ECG. Small differences in the relative timing of repolarization account for larger changes in the epicardial potentials during the T wave and, also, in the body surface T-wave amplitude. Finally, the repolarization phase of the schematic action potentials has been depicted as unrealistically slow for clarity. With a larger repolarization rate voltage differences would even be more accentuated. In summary, several repolarization gradients are responsible for the genesis of the T wave. In control dogs, and in dogs in which short-term memory was induced, there is a left ventricular apico-basal gradient with shortest repolarization times in basal areas, an antero-posterior gradient with shorter repolarization times in left ventricular anterior regions, and a right-to-left ventricular gradient with shortest repolarization times in the right ventricle [10,19,20]. In long-term memory dogs, there is also a left ventricular transmural gradient that is not present in control dogs or short-term memory dogs, with earliest repolarization times at the endocardium. Thus, relatively small regional transmural differences in epicardial repolarization time alter epicardial potential distribution which directly translates into the wave forms of the ECG [21]. No evidence was found, either in sham dogs or in memory dogs, for a midmural region with longer ARIs and RTs compared to endocardium and epicardium. Our study differs in part from previous ones on cardiac memory in that LV ARIs remained the same rather than lengthened (for review see [22]). However, the action potential recordings in the previous studies were performed on tissue slabs isolated in a perfused chamber. The reasons for the different results likely reside in the different model systems used as has been suggested by us previously [23].In that study we suggested that in tissue slabs the repolarization process is prolonged as a result of uncoupling, and reviewed data demonstrating that on going from in situ heart to wedge preparations, isolated tissue slabs and disaggregated myocytes, action potential duration becomes ever longer, especially in mid-myocardium. More recently we have confirmed that in isolated tissue the action potential duration is longer than in vivo [24], perhaps as the result of cellular uncoupling. 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