INTRODUCTION

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VENTRICULAR TACHYCARDIA (VT) and ventricular fibrillation (VF) are the major causes of ischemic-related cardiac arrhythmias, which often result in sudden cardiac death. Most ventricular arrhythmias occur during the first 10-15 min of ischemia (9) and are often due to transmural ventricular reentry (15). Although it is well known that acute ischemia in dogs depresses the excitability and velocity of conduction more rapidly in the epicardium than in the endocardium, thereby creating transmural dispersion in tissue excitability (5, 7, 8, 16, 18, 25), the mechanism by which such a response initiates reentry is only partially known.

The purpose of this study was to test the hypothesis that transmural dispersion in the response of ventricular tissue to acute ischemia caused different conduction patterns during stimulation of the epicardium compared with stimulation of the endocardium. We further hypothesized that epicardial activation could result in transmural reentry during acute global ischemia. The reasoning underlying these hypotheses is that initiation of reentry requires unidirectional block of conduction. The faster rate of depression of excitability in the epicardium compared with the endocardium could cause decremental or failed conduction along the severely depressed epicardium, but successful transmural conduction from the epicardium to the still-viable endocardium occurs when the tissue is stimulated at the epicardium during acute ischemia. This could create an opportunity for reentrant conduction from the endocardium back to the epicardium. In contrast, this transmural gradient of excitability would cause activation initiated at the endocardium to conduct relatively rapidly along the endocardium, and then slowly moving toward the epicardium, without areas of unidirectional block and therefore no reentry. Clinically, this might facilitate VT induction by a premature ventricular complex arising in the epicardium but not the endocardium.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Transmural wedges of canine ventricular muscle were prepared using procedures similar to those in our earlier studies (26, 27). In brief, we harvested hearts from adult mongrel dogs after intravenous injection of heparin sodium (5,000 units) and pentobarbital sodium anesthesia (30 mg/kg body wt) by following National Institutes of Health and institutional guidelines and quickly Langendorff perfused the hearts with an ice-cold hyperkalemia cardioplegic solution (Tyrode solution as shown below with 15 mmol/l KCl). The time interval between the heart harvest and initiation of Langendorff perfusion was <30 s. We isolated transmural wedges from the free wall of the left ventricle. Each wedge contained a section of coronary artery (which was either a branch of the left anterior descending artery or a branch of the circumflex artery, with an inner diameter of 1 mm) on the epicardial surface along the length of the wedge. The wedges were 4-7 mm wide (across the artery), 10-20 mm from the epicardium to the endocardium, and 20-30 mm along the length of the artery (27). There were 1- to 2-mm epicardial distances between the transmural recording surfaces and the perfusion artery. Two plastic cannulas were inserted into the basal and apical openings of the artery in each wedge and secured with silk sutures. The tissues were perfused with 37°C oxygenated Tyrode solution composed of (in mmol/l) 128.0 NaCl, 4.69 KCl, 1.18 MgSO₄, 0.41 NaH₂PO₄, 20.1 NaHCO₃, 2.23 CaCl₂, and 11.1 dextrose (gassed with 95% O₂-5% CO₂) through the basal cannula (in the basal opening of the artery) at an arterial pressure of 40-50 mmHg, which was monitored with a pressure transducer attached to the free end of the apical cannula. Major arterial leaks in the wedges were closed with silk sutures. The wedges were pinned to the bottom of a 37°C water jacket- warmed tissue chamber (Radnoti; Monrovia, CA) with the observation surface up (one of the transmural cut surfaces), and paced at the endocardium at a cycle length (CL) of 1,000 ms for 60 min of recovery before experimental recordings. We stained the wedges with a membrane-binding fluorescent dye, di-4-ANEPPS (Molecular Probes; Eugene, OR), at ~4 µmol/l in the Tyrode solution for ~10 min. This dye emits a membrane potential-modulated red fluorescence when it is illuminated by a green light (wavelength: 520 ± 45 nm). A 256-channel optical mapping system (with 256 photon sensors in 16 × 16 arrangement; Hamamatsu) recorded the fluorescent action potentials (AP; after a long-pass optical filter of >590 nm) in an observation area of 19.5 × 19.5 mm on the transmural cut surface at the rate of 1,000 samples · channel¹ · s¹ (Ref. 26, Fig. 1).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Schematic transmural recording surface (A) and enlarged region (B). The 16 × 16 grids in A indicate the corresponding recording regions of the photon sensors. The solid circles (sites a, b, c, d, and e) in B indicate the centers of each square region (side length: ds). Sites a, b, and c activate at time t0, t1, and t2, respectively. The curved traces (B) illustrate the isochrone lines conducting along the directions of the thin arrows.

The wedges were subjected to a preconditioning period of ischemia for 5-8 min [no irreversible damage (6, 14)] after the tissue was placed in the chamber and had been perfused with Tyrode solution for >30 min, as we have done previously (26, 27). We employed preconditioning ischemia to reduce the individual differences among the tissues in response to the inevitable ischemic insults during the process of heart removal and wedge isolation. The preconditioning was followed by >60 min of perfusion with Tyrode solution at an arterial pressure between 40 and 50 mmHg and then another episode of global ischemia of 20 min. We reduced the bath to a level lower than the top of the tissue to facilitate optical mapping during the periods of ischemia and data recording, but maintained the level to cover the tissue at all other times. We covered the opening between the optical lens of the mapping system and the tissue chamber with glass coverslips or plastic wrap as much as possible to minimize the loss of heat and moisture, whereas the electrodes and light paths were not interfered with. We visually inspected the tissues during and after each episode of ischemia and verified the moistness of the exposed surfaces of the tissues. We examined the tissue viability by the strength of muscle contraction and by the systolic arterial pressure after 1 h of recovery perfusion, by the evenness of perfusion, and by the signal-to-noise ratio and duration of the fluorescent APs. Only healthy wedges were included in RESULTS. These are the same techniques that we have employed successfully in the past (26, 27).

Isolated and arterially perfused ventricular wedges are mature experimental preparations that have been used successfully by several groups (1, 3, 21-23, 26-30). Wedges prepared and verified with the above procedures are stable. In a preparation tested previously (27), the velocity of transmural conduction slowed only ~3% during 131 min of continuous perfusion. We observed reproducible reductions in the velocity of transmural conduction during two sequential 8-min episodes of ischemia separated by 60 min of reperfusion in 12 wedges (27). We also showed previously (26) that wedges prepared with the above procedures had transmural dispersions of AP duration of ~20 and ~23 ms at pacing cycle lengths of 1,000 and 2,000 ms, respectively, which are similar to the measurements in intact ventricles (4, 10, 13).

We studied two groups of wedges. Group A contained 16 normal intact wedges. Group B contained four wedges that had the endocardium removed either chemically (in one wedge) with repeated endocardial applications of phenol solution (11) or surgically (in the other three wedges) by shaving off a 0.5- to 1.0-mm surface layer.

Using two pair of platinum bipolar electrodes, we paced the wedges continuously at a CL of 1,000 ms at the endocardium during the tissue recovery period and alternately between the endocardium and epicardium at a pacing CL of either 300 ms (11 wedges) or 600 ms (5 wedges) during the period of testing and data recording. All pacing stimuli were 2 ms in duration at twice the diastolic current threshold of activation, which was assessed at the beginning of the testing and data-recording sequences. Arterial perfusion pressure and fluorescent APs were recorded (in segments of 1.2 s) immediately before (as control) and during the second episode of ischemia (repetitively at 1-min intervals). Each recording segment was initiated 100 ms before the onset of a stimulus after 8 preceding pacing stimuli at the specified CL. Pacing continued during each recording segment. We determined the time of activation at the maximum rate of AP depolarization at each site of recording. The isochrone maps of activation time were constructed with linear spatial interpolations among the sites of observation at the specified times (26, 27). Unidirectional block of conduction is defined as the conduction that failed or was decremental along one direction, whereas it was successful along the opposite direction. Reentry is defined as a path of wave front exhibiting such unidirectional conduction and completing a circular loop.

The conduction velocities at the endocardium, subendocardium, and epicardium in each wedge (e.g., Figs. 2J and 5E) were represented by the means ± SD of the local velocities at the recording sites inside the respective regions (as indicated in Figs. 2G, 5A, and 5C). We calculated the velocities of conduction at each recording site from the times of activation at three sites, the local, a horizontal neighbor, and a vertical neighbor [e.g., times of activation t₀, t₁, and t₂ at the sites a (local), b (horizontal neighbor), and c (vertical neighbor) in Fig. 1B] with the equation
where V is the velocity of local conduction and ds is the distance on tissue surface corresponding to the centers of neighboring photon sensors (same as the side length of a square area in Fig. 1B). A single site (e.g., site a) could have multiple local velocities, depending on the available combinations of the local and two neighboring sites (e.g., a-b-c, a-d-c, a-b-e, and a-d-e for site a in Fig. 1B). Only the first endocardial row of the recording sites was used as the local sites for the calculation of endocardial velocity of conduction because Purkinje fibers are most likely covered only by the first endocardial row. The subendocardial velocities of conduction were calculated with the use of the recording sites at the second row (1.2 mm from the endocardial row) as the local sites and the endocardial row as the vertical neighbor sites. The epicardial velocities of conduction were calculated for all combinations of the neighboring sites in the first three rows from the epicardium. The means ± SD of the velocities in each wedge represented the average and heterogeneity of the velocity of conduction in the spatial resolution of 1.2 mm (i.e., the separation between neighboring sites, ds).

View larger version (53K): [in this window] [in a new window]   Fig. 2.   Typical sequences of asymmetrical conduction initiated by the epicardial (A-C and H) and endocardial (D-F and I) stimulation during acute ischemia. All stimuli (2-ms duration, twice diastolic threshold, 300-ms cycle length) were applied at time 0 to the solid circles. The numbers on the isochrone lines (A-F) indicate the time of activation (in ms). The dashed arrow traces (A-F) illustrate the direction of conduction. H and I: ischemia delayed activation at the selected epicardial (Epi), midmyocardial (Mid), and endocardial (Endo) sites along the right edge of the recording area, as well as at the epicardial (Epi-Trans) and endocardial (Endo-Trans) sites transmurally opposite to the sites of epicardial (H) and endocardial (I) stimulation. The locations of the above stimulation and observation sites are indicated in A-G. The recording area is 10 mm (transmural) × 18 mm (along the epicardium). J: means ± SD of the epicardial and endocardial velocities of conduction, initiated by the endocardial and epicardial stimulation, respectively, at the sites within the corresponding rectangular areas in G. The normalized velocities (K) are obtained by dividing the data (J) with the corresponding mean velocities at time 0 (before ischemia). L: fluorescent action potentials at the selected sites.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Figure 2 demonstrates a typical sequence of the transmural asymmetrical patterns of conduction during acute ischemia in a group A wedge. Before ischemia, activation spread rapidly in all directions from either the epicardial or endocardial site of stimulation (Fig. 2, A and D). Acute global ischemia depressed the velocity of conduction more in the epicardium than in the endocardium (Fig. 2, J and K). Although the epicardial and transmural conductions were slowed, rapid lateral endocardial conduction remained because the endocardium was less sensitive to the first 9 min of ischemia (Fig. 2, A-K). The differential rates of transmural conduction depression during acute ischemia provided the substrate for asymmetrical patterns of transmural propagation conducive to reentry. The reduced sensitivity of endocardial conduction to ischemia caused the epicardially initiated activation to conduct rapidly along the endocardium after penetrating the ventricular wall (Fig. 2, B and C). Because of less ischemia-induced depression of conduction in the endocardium compared with the epicardium and midmyocardium after >200 s of ischemia, wave fronts arrived at the right edge of the mapping area earlier in the endocardial site than in the midmyocardial site during epicardial stimulation (Fig. 2, B and C; see Fig. 2G for the locations of the sites). The epicardial site at the right edge of the mapping area was still activated earlier than the midmyocardial site up to 390 s of ischemia (Fig. 2, A and B), due to a shorter route of conduction from the epicardial site of stimulation to the lateral epicardial site than to the midmyocardial site. Therefore, epicardial stimulation initiated a wave front that arrived at the right edge of the mapping area earlier in both the epicardial site and endocardial site than in the midmyocardial site during the time window of 200-390 ms of ischemia (Fig. 2B). After 505 s of ischemia, lateral epicardial conduction became decremental (Fig. 2C), whereas conduction from the epicardial site of stimulation to the endocardium was preserved due to the gradient of increased tissue excitability along the way (Fig. 2, B and C). Because conduction along the endocardium and subendocardium was well preserved (Fig. 2, H-K), activation then spread along the endocardium and subendocardium quickly to both sides of the wedge (Fig. 2, B and C) and reentered the epicardium. The less depressed endocardial and midmyocardial regions (Fig. 2, H-K) were more capable of activating the "downstream" epicardial tissue than the neighboring severely depressed "upstream" epicardial tissue. Endocardial stimulation immediately before or after the above epicardial stimulation (Fig. 2, B and C) produced wave fronts that spread rapidly along the endocardium and then moved slowly toward the epicardium (Fig. 2, E and F) without reentry.

Figure 3 demonstrates a single loop of transmural reentry initiated by subepicardial stimulation after 801 s of global ischemia in another group A wedge. The data segment started 100 ms before the onset of a stimulus (defined as time 0, after ~20 preceding stimuli) during continued subepicardial pacing at a cycle length of 300 ms. The earliest recorded activations were at the beginning of the data segment (100 ms) at the region around site E, indicating that they were initiated by a prior stimulus applied at 300 ms or earlier. The stimulus applied at 0 ms synchronized the activation wave front to the site of stimulation (as shown by the increased separation between the isochrone lines on the epicardial side of the site of pacing). Similar to the wedge shown in Fig. 2, activation conducted transmurally to the endocardium and subendocardium but failed to conduct laterally along the subepicardium. Activation then conducted laterally in the subendocardium and reentered the epicardium, forming a closed-loop reentrant pathway. The early activation at site E reentered the same site after passing through the route along E-A-B-C-D-E and then terminated at site A.

View larger version (34K): [in this window] [in a new window]   Fig. 3.   Transmural reentry initiated by epicardial stimulation after 801 s of global ischemia. The isochrone map shows the conduction sequences of reentry. The numbers on the isochrone lines indicate the time of activation (in ms). The arrow lines indicate the directions of conduction. The wedge was stimulated at the subepicardial site (large solid circle) at 300 ms and at 0 ms. The gray region shows the epicardial conduction block. The dotted area separates the consecutive loops of reentry. The action potentials at right are recorded at the corresponding sites (A-E). The recording area is 16 mm (transmural) × 18 mm (along the epicardium).

Both the epicardial and endocardial thresholds of activation before ischemia were the same, 0.7 ± 0.3 mA, in the 16 wedges in group A. Delayed midmyocardial conduction compared with the endocardial and epicardial conduction after epicardial stimulation (e.g., along the right edge of Fig. 2B) was observed after 345 ± 166 s of ischemia in 6 of 16 group A wedges. The reentry conduction pathway from the epicardial site of stimulation to the endocardium, then laterally along the endocardium and subendocardium and back toward the epicardium (e.g., Fig. 2C), was observed in 9 of 16 wedges after 600 ± 182 s of ischemia. Reentry continued for >1 loop in four of nine wedges after 708 ± 230 s of ischemia at CLs of 328 ± 106 ms. Endocardial stimulation, applied immediately before or after the epicardial stimulation that initiated reentry, caused activation that conducted rapidly along the endocardium and then moved slowly to the epicardium without reentry (e.g., Fig. 2, E and F) in six of the above nine wedges. We observed unidirectional block of conduction [similar to our previous observations (27)] at the border of the regions having different rates of responses (i.e., 1:1 endocardial and 2:1 and/or 4:1 epicardial responses) in response to the endocardial stimuli during acute ischemia in eight wedges among a total of 11 wedges that were paced at a CL of 300 ms.

Figure 4 shows the statistics and correlations of the ischemia-induced depression in the velocity of conduction in the endocardium (13 wedges), in the subendocardium (13 wedges), and in the epicardium (14 wedges) in group A. Before ischemia, the velocity of conduction in the endocardium was faster than in the subendocardium (P = 0.01) and than in the epicardium (P = 0.00, Fig. 4A). The faster endocardial conduction could be the contribution of Purkinje fibers. The velocity of conduction in the subendocardium and epicardium were similar (P = 0.07) before ischemia (Fig. 4A). Eight minutes of global ischemia depressed the velocity of conduction significantly (P = 0.01, 0.02, and 0.00, for the endocardium, subendocardium, and epicardium, respectively) and enhanced the transmural dispersion of the velocity of conduction, resulting significant differences among the endocardium, subendocardium, and epicardium (Fig. 4A). Eight minutes of ischemia depressed the velocity of conduction in the epicardium to 34% of the velocity before ischemia, significantly less than in the endocardium (84%, P = 0.00) and subendocardium (79%, P = 0.00, Fig. 4B). The depression of conduction velocity in the endocardium and subendocardium were similar after 8 min of ischemia (P = 0.41, Fig. 4B). The transmural dispersion in the ischemia-induced depression of conduction velocity provided the substrate for the epicardially initiated reentry and transmural conduction asymmetry as we demonstrated above, as well as provided further detail to support and extend the previous observations that ischemia depressed the activation and conduction more in the epicardium than in the endocardium (8).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   The velocities of conduction before and after 8 min of ischemia in the group A wedges. A: average and standard deviation of the endocardial (Endo), subendocardial (S-Endo), and epicardial (Epi) mean velocities of conduction in the wedges. The mean velocities of conduction in each wedge are calculated from the local velocities at the recording sites in the endocardial row, in the second endocardial row (1.2 mm from the endocardial row), and in the first three epicardial rows, respectively, as stated in METHODS. B: depression in the velocity of conduction after 8 min of ischemia in the wedges. The depression in the velocity of conduction in each wedge is obtained by dividing the mean velocities after 8 min of ischemia with their corresponding values before ischemia. Error bars indicate SD. The numbers in parenthesis indicate the number of wedges. The thin double-arrow lines and the numbers above show the statistical correlations between the pairs of data (paired Student's t-test).

The above transmural asymmetric pattern of conduction and reentry (e.g., Figs. 2 and 3) were not observed in four separate wedges (group B) that had the endocardium removed either surgically or by phenol application. Both epicardial and endocardial stimulation initiated similar patterns of transmural conduction centered at the sites of stimulation in these four wedges (e.g., Fig. 5). This result supported the previous observations by Gilmour and Zipes (7) that Purkinje fibers contributed to the transmural heterogeneities in tissue excitability during acute ischemia.

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Conduction in a group B wedge that had its endocardium (0.5- to 1-mm surface layer) removed. Subepicardial (A and C) or endocardial (B and D) stimuli were applied to the dotted sites at 0 ms. The numbers on the isochrone lines indicate the times of activation (in ms). The S-Endo and Epi velocities of conduction (means ± SD) in E are calculated from the activation times at the sites within the corresponding rectangular areas in A and C. Depression in the velocities of conduction after 601 s of ischemia (F) is derived from E by dividing the data of after ischemia with their corresponding mean velocities before ischemia. The recording area is 13.4 mm (transmural) × 16 mm (along the epicardium).

The movies corresponding to Figs. 2C, 2F, and 3 can be viewed at http://ajpheart.physiology.org/cgi/content/full/283/5/H2008/DC1.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

New observations. This study demonstrated the existence of transmural dispersion in the velocity of local conduction before ischemia in isolated canine ventricular wall. Acute ischemia enhanced the transmural dispersion with more rapid depression of excitability and conduction in the epicardium than endocardium. This differential response permitted activation initiated in the epicardium to block laterally in the epicardium, conduct transmurally to the endocardium, travel laterally in the endocardium, and return to the epicardium to complete reentrant loops. Endocardial stimulation, applied immediately before or after the above epicardial-initiated reentry, initiated activation that conducted along the endocardium rapidly toward the epicardium without reentry. Removal of the endocardium prevented the above patterns of asymmetrical conduction and reentry during epicardial stimulation. On the basis of these observations, we conclude that ischemia-induced transmural gradient of excitability provided the substrate for reentry during epicardial stimulation.

Previous studies. The mechanism of unidirectional block of conduction and transmural reentry initiated by epicardial stimulation, as shown in this study, is based on the ischemia-induced transmural gradient of tissue excitability, in contrast to the ischemia-induced transmural gradient of refractory period during endocardial stimulation that was the mechanism responsible for the reentry in our previous study (27). We reported previously (27) that the ischemia-induced transmural dispersion in tissue refractoriness and the tissue heterogeneity could block conduction unidirectionally during endocardial stimulation and cause reentry. We observed that endocardial activation could fail to conduct directly to the epicardial and midmyocardial regions where the local refractory periods were longer than the endocardial pacing cycle lengths (300 ms), resulting 2:1 and/or 4:1 responses to the pacing. However, the impulse could conduct 1:1 to other regions having refractory periods less than the pacing cycle length. Activation then conducted from the epicardium transmurally through the regions of 2:1 and/or 4:1 responses and reentered the subendocardium, where activation was originally initiated, forming reentry. We observed similar phenomena of multiple zones of tissue responses to pacing stimuli in the present study. Both our current and previous studies are complementary because acute ischemia-induced both transmural gradients in the tissue excitability and in the tissue refractory period. The spectrum of tissue heterogeneity and responses to acute ischemia provided the substrates for both types of reentry to occur when cardiac muscle was stimulated at the right time and at the right location (epicardium or endocardium).

Limitations. We used the wedge preparations and global ischemia protocol to focus on the contributions of the ischemia-induced depression to the initiation of reentry and to eliminate other factors not included in the scope and hypothesis of this study, e.g., the border zone of regional ischemia, which occurred clinically when a single branch of the coronary artery was blocked. Regional ischemia is a complex phenomenon that includes both the heterogeneous perfusion (the perfused and ischemic regions and the border zone) and the local responses of tissue to ischemia. The wedge preparations also limited the possible patterns of conduction. However, the contributions of transmural gradient of tissue excitability to the unidirectional block of conduction and initiation of reentry during acute global ischemia as we demonstrated in the wedges should also exist in intact heart.

Clinical implications. Normal cardiac tissue is heterogeneous (2) and its response to ischemia is even more so. The combination of tissue heterogeneities and transmural gradients of the tissue excitability and refractory period create multiple possibilities for reentry to occur during acute ischemia. Thus, depending on the nature of the heterogeneity, premature ventricular complexes originating in the epicardium and/or in the endocardium could precipitate reentry. The epicardially initiated reentry during global ischemia as we demonstrated here is a component contributing to the clinical VT and VF during acute ischemia. The understanding of the possible mechanisms of reentry initiation will provide the foundations for further research on the prevention and management of ischemia-induced VT and VF.