Profibrillatory Effects of Intracoronary Thrombus in Acute Regional Ischemia of the In Situ Porcine Heart
Background An intracoronary thrombus during regional ischemia is related to life-threatening arrhythmias. The electrophysiological consequences of a thrombus are unknown.
Methods and Results In open chest pigs, regional ischemia was induced by intracoronary injection of a thrombus (protocol 1). In protocol 2, coronary ligation was followed by injection of heparinized blood. Three consecutive episodes of ischemia (10 minutes) and reperfusion (20 minutes) were studied in protocols 3 and 4 (ligation). During the former, an intracoronary thrombus started the third period of ischemia. Multiple (78) local electrograms were recorded simultaneously, and activation patterns were determined. In a first period of ischemia, ventricular fibrillation (during the first 10 minutes) occurred more often after intracoronary thrombosis than during the other protocols (4/7 versus 2/19, P<.05) despite similar size of the ischemic tissue. The incidence of delayed arrhythmias (between 15 and 30 minutes) was not different. Epicardial activation delay was larger 2 to 4 minutes after intracoronary thrombosis compared with ligation. ST elevation was larger with than without a thrombus (2 minutes of ischemia, 12.9±4.1 versus 8.2±3.0 mV; ±SD, P<.05). In protocols 3 and 4 the second period and third period of ischemia were similar irrespective of the presence of an intracoronary thrombus.
Conclusions More conduction slowing underlies the profibrillatory effect of an intracoronary thrombus relative to coronary ligation. After preconditioning with ischemia, the profibrillatory effects are no longer detectable.
Acute obstructive coronary artery thrombosis in patients results in life-threatening arrhythmias.1 The mechanisms underlying these arrhythmias have been studied in animal models in which one of the coronary arteries is cross-clamped or ligated. Arrhythmias observed during ischemia produced in these animal models resemble those in patients.1
Although the ischemia-induced electrophysiological changes in ischemia can be simulated by perfusing tissue with hypoxic, hyperkalemic and acidotic Tyrode’s solution,2 the consequences are much more severe when myocardium is superfused with blood drained from ischemic tissue with the same combination of hypoxia, hyperkalemia, and acidosis.3 This has inspired the proposal of unknown factors released from ischemic tissue. Products of altered lipid metabolism (for example, lysophosphatidylcholines [LPCs]) have been implicated as a potential factor.4
Recently, intracoronary thrombosis was described to cause a greater incidence of life-threatening ventricular arrhythmias than ischemia produced by balloon occlusion, despite similar magnitudes of ischemic myocardial mass.5 In that study a thrombus was allowed to grow inside the coronary artery and therefore a period of low-flow ischemia preceded total ischemia. This study was undertaken to study the electrophysiological mechanism of the proarrhythmic effect of an intracoronary thrombus in a blood-perfused animal model with poor collateral circulation. In particular, we aimed to document the arrhythmogenic effect without preceding low-flow ischemia and to differentiate between early and late ventricular arrhythmias.6 We studied local electrograms and the pattern of activation of the ischemic tissue in the course of 30 minutes of regional ischemia and at the onset of ventricular fibrillation (VF) in pigs with and without an intracoronary thrombus. Because LPCs accumulate during ischemia even in the absence of thrombosis and cause structural changes in the sarcolemma,4 we hypothesized that the arrhythmogenic effect of intracoronary thrombosis is diminished after ischemic “preconditioning.” Therefore, we also studied the effect of an intracoronary thrombus after preconditioning.
This study provides evidence that increased conduction slowing underlies the occurrence of early VF in animals with intracoronary thrombosis compared with animals with cross-clamping of the coronary artery. The electrophysiological effects of intracoronary thrombosis are lost after two reversible periods of ischemia and reperfusion.
The experiments were in accordance with the institutional guidelines for the handling and care of animals. Pigs (weight, 15 to 20 kg) were premedicated with azaperon (2 mg/kg, Stresnil, Janssen), ketamine (15 mg/kg, Nimatek), and atropin (0.5 mg, Centrafarm) intramuscularly. Pentobarbital (15 mg/kg, Nembutal) was administered intravenously. After tracheal intubation the animals were artificially ventilated with an infant ventilator (Loosco) with a 1:1 mixture of O2 and N2O2. The carbon dioxide content of the expiratory gasses was continuously monitored. A continuous venous drip, also used to administer drugs, was introduced in a peripheral vein. A standard ECG was recorded (Datex) on a high speed chart recorder (Graphtec). A cannula was inserted into the right carotid artery and connected to a pressure transducer (Datex Cardiocap, Datex). Rectal temperature was recorded and could be maintained between 36° and 37°C with a thermostatically controlled blanket.
The thorax was opened through a midsternal incision. A pericardial cradle was constructed. Two ligatures were passed underneath the left anterior descending artery (LAD) just below the first diagonal branch. An epicardial stimulation electrode was attached to the left atrium.
A 30-second LAD occlusion was made to identify the cyanotic border. An epicardial array (13×6; interelectrode distance, 4 mm) of wick electrodes was attached to the epicardium with thin (4–0) atraumatic sutures. The multielectrode covered the prospective ischemic tissue but allowed a clear view of the artery. An bipolar hook electrode was inserted in the left ventricular free wall (not affected by coronary ligation) close to the basal corner of the multielectrode. After instrumentation, no interventions were performed for at least 30 minutes.
Anesthesia was maintained with α-chloralose (0.2 g per 30 minutes, Merck) and a continuous infusion of sufentanil (25 μg/h).
The left atrium was stimulated with rectangular current pulses (2-month duration, twice diastolic threshold, cycle length 500 ms). After a delay of 40 to 80 ms, the left ventricle was stimulated with a rectangular current pulse (1 ms, twice threshold). This produced a wide QRS complex without a fall in blood pressure. In the case of VF, the heart was defibrillated (Hewlett-Packard 7802 D) with a countershock applied to the epicardial surface. If more than 6 shocks were necessary, the experiment was discontinued.
Seventy-eight epicardial electrograms were simultaneously DC-amplified (×32) and digitized with a sample frequency of 1 kHz. Eight selected electrograms were continuously written on a chart recorder and displayed on a monitor. Digitized recordings had a duration of 1.8 seconds and were made at 30-second intervals in the first 3 minutes of ischemia and at 1-minute intervals at later stages of ischemia or when arrhythmias occurred. Data were stored on a hard disk of a PDP 11/73 computer for later analysis (activation patterns, morphology of the electrograms). Local activation times were defined as the moment of the steepest negative deflection and were determined automatically. The signals were visually examined for signs of local inexcitability showing as a monophasic electrogram.3
Epicardial conduction was quantified by calculating the difference between the moments of earliest activation (nonischemic tissue) and latest activation (ischemic tissue) of a ventricular paced beat. An activation delay between two adjacent sites greater than 30 ms (corresponding to an apparent conduction velocity of 13 cm/s) was arbitrarily defined as conduction block. This delay was calculated for all neighboring electrode sites except when sites were inexcitable. The number of sites with conduction block was counted at selected moments of ischemia.
Local ST-elevation was measured relative to the TQ-segment at 170 ms after the stimulus artifact and the maximum potential difference between the TQ and the ST segment of all electrode sites after 2 minutes and 5 minutes of ischemia (maximum ST elevation) was determined. The different amount of shift after the introduction of a thrombus or heparinized blood between the animals precluded the measurement of amount of TQ-depression (relative to preocclusion values).
Arrhythmias were defined according to the Lambeth conventions.7 Ischemia-induced ventricular arrhythmias were divided into early (up to 10 minutes) and delayed (15 to 30 minutes) arrhythmias (protocols 1 and 2). The interval between 10 and 15 minutes of ischemia was arrhythmia free in all animals.
The electrophysiological border was defined as the line separating tissue with ST elevation and with ST depression.
Four study groups were formed
1. First (prolonged) occlusion + thrombus (7 pigs): The LAD was cannulated just distal to the prospective site of occlusion and was then ligated. A fresh thrombus (2 mL) was injected and appeared in the distal branches of the artery. The thrombus was prepared by mixing 2.3 mL of the pig’s blood with 0.2 mL of thrombin (300 U) in saline in a syringe 1 minute before the moment of injection. The ionic composition of the resulting prothrombotic solution was not significantly different from normal blood. The syringe was kept at 37°C in a water bath. The experiment was stopped after 30 minutes of ischemia or when defibrillation failed.
2. First (prolonged) occlusion, sham (4 pigs): The LAD was cannulated as above. The same protocol was followed with the exception that 2 mL of heparinized blood was injected.
3. Repetitive ischemia + thrombus (6 pigs): The LAD was cross-clamped with an atraumatic vessel clamp for 10 minutes. After that, a reperfusion period of 20 minutes’ duration was allowed. These periods of ischemia served as control for the first 10 minutes of ischemia in protocol 1. A second similar cycle of ischemia and reperfusion was followed by a third period of ischemia (10 minutes) initiated by injection of a thrombus (see under paragraph 1). The second period of ischemia served as a control for the third.
4. Repetitive ischemia, control (9 pigs): To confirm that under our experimental conditions the second and third period of ischemia are the same,8,9 three consecutive periods of ischemia (10 minutes) were made separated by 20 minutes of reperfusion by cross-clamping the LAD. The first periods of ischemia also served as a control for protocol 1.
If VF occurred the heart was internally defibrillated and the acquisition of data were stopped but the artery was left occluded until 10 minutes of ischemia. In protocols 3 and 4, all parameters had returned to preischemic values after 20 minutes of reperfusion. In protocol 3, one experiment had to be discontinued because of the occurrence of VF that could not be terminated (see above).
The average time between cannulation and injection of the thrombus (protocol 1 and 3) or of blood (protocol 2) was 12.6±3.0 seconds (mean±SEM) and was not different between groups. At the conclusion of the experiment, the heart was extirpated, blotted dry, and weighed after removal of the atria. Then the heart was perfused with a solution of indian ink in saline. The ventricular tissue with a black discoloration was dissected and weighed. The ratio between the two masses served as an index of the relative size of the ischemic zone.
Differences between proportions were tested with the Fisher’s exact test. The critical level for significance was assumed P=.05. ANOVA followed by the Newman Keuls post hoc test was used for multiple comparisons. The Student’s t test was used for the comparison of (pairs of) data. Data are presented as mean±SD unless indicated otherwise.
In the control experiments of protocol 2, early VF (during the first 10 minutes of ischemia) did not occur (0/4 hearts). This was not significantly different from the occurrence of early VF (2/15 hearts) during the first periods of ischemia in the hearts from protocol 3 and 4 (repetitive ischemia). Therefore, these control experiments were pooled (VF in 2/19 hearts, 11%). When a thrombus was introduced in the LAD early VF occurred in 4 of 7 (57%) hearts (P<.05 versus control hearts). The onset of early VF was 3 minutes 19±44 seconds (mean±SEM) in the group with intracoronary thrombus (protocol 1). In the control group VF occurred after 3 minutes, 55 seconds and 4 minutes, 10 seconds.
To investigate whether the increased incidence of VF was associated with a larger number of ventricular premature beats, doublets or tachycardias these arrhythmias were counted during the first three minutes of ischemia. They were not significantly different between the groups (not shown).
In one of the experiments of protocol 1 (intracoronary thrombus), the delayed phase of arrhythmias was not reached because of technical problems. VF occurrence in the delayed phase was not significantly different between the study groups (3/6 versus 4/4, protocols 1 and 2, respectively). In the animals with an intracoronary thrombus, the onset of delayed VF ranged between 19 minutes and 27 minutes and in the sham group between 18 minutes and 28 minutes.
The relative size of the ischemic zone was not different between the hearts from protocol 1 and 2 (32.1±0.6% versus 27.9±2.8%, mean±SEM). Also, the fall in systolic pressure after 2 minutes of ischemia was not different (group 1, 9±3.6 mm Hg; group 2, 7±3 mm Hg).
Fig 1⇓ shows signals recorded from the normal and ischemic epicardium after 2.5 minutes of cross-clamping the LAD (top), and in another heart in which a thrombus was introduced into the LAD (bottom). In the ischemic zone, electrograms with maximum ST-elevation were selected. In the heart without the intracoronary thrombus, no VF occurred during the first 10 minutes of ischemia. Although the relative size of the ischemic zone was larger in the control heart (30%) than in the “thrombus” heart (18%) the electrophysiological signs of ischemia were more severe in the latter. The TQ segment was more depressed and the ST segment more elevated (ischemic zone) and the ST alternation between subsequent beats more pronounced in the heart with the thrombus. Note that the activation that initiated VF demonstrated an initial negative deflection (superimposed on a T wave) and preceded activity in the ischemic tissue. It appears to originate from the normal tissue, a sequence not different from that in control ischemia.1
In acute ischemia, the onset of VF correlates with the occurrence of (alternation of) deeply negative T waves.10,11 The moment at which a negative T wave or its alternation was detected at any electrode was after 2.5±0.5 minutes of ischemia in the control experiments (n=15) and 2±0.2 minutes in the “thrombus” experiments (P<.01). This suggests that the time course of ischemia is slightly accelerated in ischemia produced by an intracoronary thrombus.
Fig 2⇓ shows maps of the distribution of ST elevation (relative to the diastolic potential, arrows) after 2 minutes of ischemia in a heart without and in a heart with an intracoronary thrombus (top and bottom respectively). The potential difference was measured 170 ms after the stimulus artifact. Ischemia in the presence of an intracoronary thrombus is associated with a higher maximum ST elevation than ischemia caused by ligation alone. Maximum ST elevation after 2 minutes of ischemia was 8.2±3.0 mV in the control experiments (n=15) and 12.9±4.1 mV in the 7 hearts with an intracoronary thrombus (P<.05). After 5 minutes of ischemia, maximum ST elevation was 15.4±5.1 (n=13) and 22.8±6.0 mV (n=4, P<.05). In either group, ST elevation measured after 150 or 190 ms was not different from ST-elevation 170 ms after the stimulus artifact.
Fig 3⇓ shows activation patterns of the myocardium underlying the electrode grid (dots in the left upper panel) after 2 and 5 minutes of ischemia in an experiment with coronary cross-clamping (left) and with an intracoronary thrombus (right). The position of the border between ischemic and normal tissue (bold line) is the same in the two hearts. After 2 minutes of ischemia activation time of the tissue under the electrode is about 30 and 70 ms in the control heart. and in the heart with a thrombus, respectively. The sites with an interelectrode activation delay of more than 30 ms, indicated by small bars, are more manifest in the thrombus heart than in the control heart. The differences between the two hearts have disappeared after 5 minutes of ischemia (bottom).
Fig 4⇓ shows the averaged changes of the longest activation delay of the tissue underlying the electrode (left) and of the number of sites with an activation delay of more than 30 ms (right) during the first 10 minutes of ischemia in experiments with an intracoronary thrombus (protocol 1) and in all experiments with a first period of ischemia without an intracoronary thrombus (protocols 2, 3, and 4). The differences in activation time between the two groups were statistically significant from 2 until 4 minutes of ischemia (ANOVA, P<.05). The differences in the number of sites with activation block did not reach statistical significance. However, the maximum activation delay between adjacent sites was 52±8.7 ms in the hearts with an intracoronary thrombus and 25.5±11.4 ms in control experiments after 4 minutes of ischemia (t test, P<.01).
The initiation of VF was captured in two hearts with an intracoronary thrombus. The first beat of VF always originated from the normal side of the ischemic border.
In the animals belonging to protocol 3 (n=5) VF occurred two times during the second episode of ischemia and 2 times during the third period (with a thrombus). In the animals subjected to protocol 4 (repetitive reversible ischemia, n=9) VF occurred twice during the second and 4 times during the third period of ischemia (not significantly different). Also between the groups 3 and 4 the occurrence of VF was not significantly different during the third ischemic episode (2/5 versus 4/9, with and without a thrombus, respectively).
VF occurred during the first ischemic period in 2 of 15 animals in the pooled groups 3 and 4. This was not significantly different from VF in the second period of ischemia in these groups (5/15).
In protocol 3 the third period of ischemia was initiated by the introduction of a thrombus in the LAD. The electrograms recorded from the ischemic tissue did not demonstrate significant differences between ischemia produced by cross -lamping the artery (second period of ischemia) and ischemia produced by an intracoronary thrombus. In the 5 animals subjected to protocol 3 maximum change of the ST segment (after 2 minutes of ischemia, relative to the TQ segment) was the only statistically significant alteration difference between the second and the third episode of ischemia (5.8±0.4 versus 3.4±0.9 mV, respectively). No differences were detected in the time to alternation (3.6±0.5 versus 3.7±0.7 minutes) in the second versus the third period of ischemia.
The second and third period of ischemia are the same in terms of the electrophysiological and ionic changes.8,9 In nine animals, three episodes of repetitive periods of ischemia were produced (protocol 4) to serve as a control group for protocol 3. Indeed, in the hearts of protocol 4 no statistically significant differences were detected between the electrophysiological changes during the second and the third episode of ischemia. Neither were there any statistically significant differences between the changes during the third period of ischemia in hearts of protocol 3 and during the third period of ischemia in hearts of protocol 4 (data not shown).
The activation patterns of hearts from protocol 3 in which the introduction of an intracoronary thrombus initiated the third period of ischemia were not different after the same duration of ischemia between the second and the third period of ischemia. In Fig 5⇓, the averaged activation times of the tissue underlying the electrode is plotted during the two periods of ischemia. No statistically significantly differences were detected. The number of sites with an activation delay of more than 30 ms relative to their neighbors was identical between the two periods of ischemia.
This study shows that the presence of an intracoronary thrombus is associated with an increased occurrence of VF and with more conduction slowing during the first minutes of ischemia. The appearance of delayed VF was not different between hearts with an intracoronary thrombus and with coronary cross-clamping. Initiation of VF requires the simultaneous occurrence of a premature beat and a suitable “substrate.”12 The number of premature beats, doublets and ventricular tachycardias within the first 3 minutes of ischemia was not different between the hearts with and without an intracoronary thrombus. Therefore, the increased incidence of VF must be accounted for by a more favorable “substrate” formed by more conduction slowing. A premature activation generated immediately after repolarization encounters only partially recovered tissue especially in the ischemic myocardium,13,14 resulting in even more marked slowing of conduction. Also, ST elevation was larger in hearts with than without an intracoronary thrombus. ST-segment elevation relative to the diastolic potential is a combined measure of ischemia induced resting membrane depolarization and of loss of local action potential amplitude and duration.10
In two cases the start of VF was recorded and activation mapping was performed. In both cases the initiating beat appeared to originate from the normal side of the ischemic border. This is not different from the start of VF during ischemia caused by LAD cross-clamping.11 Although three-dimensional activation mapping is required for the identification of the definitive origin of the first beat of VF15 the similarity of the observations on the epicardial surface in hearts with and without an intracoronary thrombus underscore that not the initiating beat but the underlying electrophysiological changes (the “substrate”) explain the increased incidence of VF in hearts with an intracoronary thrombus.
Our results corroborate a study by Goldstein et al,5 who observed an increased incidence of VT/VF in a dog model in which an intracoronary thrombus formed spontaneously compared with animals with coronary balloon occlusion. In their model the onset of ischemia was defined when coronary blood flow became zero and total occlusion of the artery was preceded by a period of decreasing flow. As a consequence of the ill-defined onset of ischemia, no distinction between early and delayed ischemia induced ventricular arrhythmias could be made, nor between the associated electrophysiological mechanisms.6 Moreover, the arrhythmogenic effect of the thrombus may have been underestimated,5 because total ischemia preceded by flow reduction (Harris’ two-stage occlusion16) is antiarrhythmic,17 probably because dangerous intermediary levels of extracellular potassium are less prevalent.9 In the study by Goldstein et al,5 the period of decreased flow was absent in the control group.
In our study the electrophysiological effects of the thrombus lasted for only several minutes, which is compatible with the rapid release of LPCs from endothelium and myocardium and their accumulation in ventricular myocytes after stimulation with a thrombin receptor activating peptide.18–20 The effects of thrombin may have been more prolonged but conduction slowing may have been dominated by the effects of progressing ischemia, when the extracellular rise of potassium concentration results in membrane depolarization with consequent conduction slowing and decreased excitability.21,22 The precise mechanism by which thrombin exerts its electrophysiological effects is not known, but several factors may contribute. Activation of the thrombin receptor leads to a larger increase in intracellular Na+,23 to increased Ca2+ entry in embryonic myocytes24 and cultured neonatal rat myocytes,25 and causes increased automaticity and prolonged repolarization in Purkinje fibers.26 The association between the effects of thrombin and the release of LPCs suggests a causal relation.18,20,23
In fact, LPCs do accumulate in ischemic myocardial tissue4,27,28 and LPCs administered to normal myocardium produce effects similar to ischemia, especially when pH is lowered.3,27,29 Indeed, LPCs and other amphiphiles4 slow recovery from inactivation of Ina,30 increase intracellular [Na+],31 increase cytosolic Ca2+,32 may cause delayed after depolarizations,31 induce resting membrane depolarization,33 and closely reproduce the electrophysiological changes seen in ischemia.29,34
In cats with VF during regional myocardial ischemia the LPC content of the ischemic myocardium was larger than in animals without arrhythmias.28 Also, inhibition of the accumulation of LPCs during a 5-minute period of coronary occlusion prevented early occurrence of ventricular tachycardia and VF.35
Although LPCs are produced in ischemic heart after heparinization,36 their release from endothelial cells18 and from ventricular myocytes is stimulated by thrombin.19,20 Suggestions have been made about the intracellular signaling pathway of thrombin receptor activation.5 The purpose of the present study was to investigate the electrophysiological mechanism of the effects of thrombin rather than to study the pathways of action.
We have chosen a protocol of cycles of 10 minutes of ischemia and 20 minutes of reperfusion because at the end of a cycle all parameters had returned to their preischemic value.37 The first period of ischemia has different electrophysiological consequences compared to any after period of ischemia.8,9 Fleet et al8 have studied the ionic and electrophysiological changes in sequential periods of ischemia and reperfusion, and reproducibility was attained after the second or third period of ischemia. This study confirms that a second period of ischemia can be used as a control for a third period. Both longer (30 minutes) and shorter (15 minutes) periods of reperfusion preceded by 10 minutes of ischemia have been shown to precondition hearts.38,39
An intracoronary thrombus in the third period of ischemia did not have electrophysiological consequences different from those during the second occlusion. This observation can be explained by a larger rise of [K+]out during the (second and) third than the first ischemic episode.8,40 The high [K+]out could mask the effects of the intracornary thrombus on conduction. Indeed, more sites with activation block are present after the second and third occlusion than after the first occlusion (Figs 4⇑ and 5⇑). Alternatively, the effects of LPCs are particularly evident when tissue pH is low27 but pH change during subsequent periods of ischemia decreases8,40,41 and may thus decrease the impact of the LPCs. A third mechanism for the absence of an effect of an intracoronary thrombus in the third ischemic episode may be operative. In the original report that set off the search for the ischemia induced factor(s) involved in arrhythmogenesis, heparinized blood was collected from the ischemic tissue.3 The effects of the “ischemic” blood on superfused myocardium were more severe than expected based on the combination of hyperkalemia, hypoxia, and acidosis alone. Obviously, in that study the unknown factor(s) must have been present without the involvement of activation of the coagulation system. Products of lipid metabolism have been suggested as a source of these unknown factors.4 We speculate that in our study amphipathic metabolites are incorporated in the sarcolemma4 during the first period of ischemia even in the absence of activation of the coagulation system and are not washed out during the subsequent period of reperfusion. In this manner ischemic preconditioning may mimic the electrophysiological effects of an intracoronary thrombus. This effect may not be fully appreciated from our studies because the occurrence of VF in the second period of ischemia, although larger, was not significantly different from the the first period of ischemia (without a thrombus). This may have resulted from the concurrent alterations in the change of [K+]out and pH (as discussed above) which is different in the second from the first period of ischemia as well.37,40 In the second and the third episode of ischemia, however, the rise in [K+]out is the same.8,9 In the same model as ours later episodes of regional ischemia are associated with an increasing incidence of VF.8
The influence of intracoronary thrombosis underscores the necessity of the coincidence of a “trigger” with an optimal “substrate.”12,42 A thrombus advances the onset of the suitable “substrate” in time, thereby increasing the likelihood of a premature beat resulting in VF. A thrombus is profibrillatory in hearts not protected by ischemic preconditioning. This may at least in part explain the clinical observation that sudden obstruction of the coronary artery in patients without preexisting narrowing of the coronaries, which often occurs in young patients, is associated with a higher incidence of primary VF than in older patients with preexisting coronary stenoses, despite similar sizes of the infarcted area.43
The authors gratefully acknowledge the expert biotechnical support given to this study by C. Belterman.
- Received June 3, 1997.
- Revision received August 19, 1997.
- Accepted August 27, 1997.
- Copyright © 1997 by American Heart Association
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