REPV vulnerability and Shortening of Operational Period

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1 Mechanism of Acceleration of Functional Reentry in the Ventricle Effects of ATP-Sensitive Potassium Channel Opener Takumi Uchida, MD; Masaaki Yashima, MD; Masamichi Gotoh, MD; Zhilin Qu, PhD; Alan Garfinkel, PhD; James N. Weiss, MD; Michael C. Fishbein, MD; William J. Mandel, MD; Peng-Sheng Chen, MD; Hrayr S. Karagueuzian, PhD Background The effect of effective refractory period (ERP) shortening on the vulnerability and characteristics of induced functional reentry in the ventricle remain poorly defined. We hypothesized that ERP shortening increases ventricular vulnerability to reentry and accelerates its rate, as is the case in the atrium. Methods and Results The epicardial surfaces of 19 isolated and superfused canine right ventricular slices (4 4 cmand 2 mm thick) were mapped with 480 bipolar electrodes 1.6 mm apart. Vulnerability was tested during pacing at a cycle length (CL) of 600 ms and with a single premature stimulus of 5-ms duration at increasing current strength of 1 to 100 ma. Cromakalim (10 mol/l), an ATP-sensitive potassium channel opener, caused a significant (P 0.001) shortening of the ERP but had no effect on conduction velocity. Cromakalim increased (P 0.01) the vulnerability (product of current and the stimulus coupling interval) for reentry induction. Reentry had a significantly shorter CL and lasted for a longer duration (P 0.001). The central core around which the wave front rotated became smaller, which caused shortening of the CL of reentry. A significant (P 0.001) linear correlation was found between core size and reentry CL. These effects of cromakalim were reversible. Two-dimensional simulation studies using the modified Luo-Rudy I model of cardiac action potential, in which the refractory period was variably shortened by a progressive increase of the time-independent potassium conductance, reproduced the experimental findings. Conclusions ERP shortening by an ATP-sensitive potassium channel opener increases ventricular vulnerability to reentry and accelerates its rate by decreasing the core size around which the wave front rotates. (Circulation. 1999;99: ) Key Words: reentry tachycardia potassium cromakalim waves The role of ATP-sensitive potassium (K-ATP) channel openers in the genesis (vulnerability) of ventricular reentrant wave fronts remains undefined. 1 Although this class of agents shortens action potential duration (APD) and the effective refractory period (ERP), 1 3 as in myocardial ischemia and hypoxia, 4 the influence of these agents on ventricular vulnerability to functional reentry (spiral wave) 5 formation by an S 2 stimulus remains poorly explored, as do the characteristics of the induced spiral wave. Spiral-wave theory predicts that shortening of the ERP shortens the period of the spiral wave by decreasing the core size around which rotation occurs. 6 In accordance with this theory, atrial tissue studies 7,8 showed that acetylcholine-induced shortening of the ERP accelerates the rate of functional reentry, increases the vulnerability of the atrium to a premature stimulus, and increases the duration of the induced activity. In the present study, we hypothesized that shortening of the ERP by cromakalim, a K-ATP channel opener, would increase ventricular vulnerability to reentry and accelerate its rate. Methods Tissue Preparation All experiments were performed in accordance with US Public Health Service guidelines for the care and use of laboratory animals. Seventeen mongrel dogs of either sex weighing between 23 and 28 kg were anesthetized with sodium pentobarbital 35 to 40 mg/kg IV. The hearts were removed through a midsternal approach and placed in cold, oxygenated Tyrode s solution An epicardial sheet 2 mm thick and cm wide was taken from the right ventricle as described previously. 9,11 The epicardial sheets were then mounted in a tissue bath either face down 9 or face up for action potential recordings (see below). 9,10 Excitability and Vulnerability Measurements Bipolar electrodes 2 mm apart (silver silver chloride) were used to pace the tissues. Regular stimuli (S 1 ) at a cycle length of 600 ms and of 5 ms duration with increasing current strength starting at 0.1 ma were applied until 1-to-1 capture occurred. This current defined the diastolic excitability current threshold (DET). To induce reentry and generate strength-interval plots, 8,9 a premature (S 2 ) stimulus (similar electrode as the S 1 ) with increasing current strengths (1 to 100 ma) Received April 15, 1998; revision received September 15, 1998; accepted September 25, From the Division of Cardiology, Burns and Allen Research Institute, Cedars-Sinai Medical Center, and the Department of Medicine, Division of Cardiology, UCLA School of Medicine, Los Angeles, Calif. Correspondence to Hrayr S. Karagueuzian, PhD, Cedars-Sinai Medical Center, Davis Research Bldg, Room 6066, 8700 Beverly Blvd, Los Angeles, CA [email protected] 1999 American Heart Association, Inc. Circulation is available at 704

2 Uchida et al February 9, TABLE 1. Ventricular Excitability and Vulnerability Parameters S 1 S 2 Interval, ms Current Strength, ma DET, ma ERP, ms Inner Outer LLV ULV Control Cromakalim P NS NS Inner and Outer indicate shortest and longest regular drive premature stimulus (S 1 S 2 ) coupling intervals that induce reentry, respectively; LLV and ULV, lower and upper limits of vulnerability, respectively. Data are mean SD of 9 tissues. Measurements were made during regular pacing at 600-ms cycle length. and decreasing coupling intervals was applied 1.5 to 2 cm distal to the S 1 site along the long axis of the fiber orientation. Determination of the Excitable Gap During reentry, trains of stimuli (2 to 5 ma) were given at the S 1 site, and action potentials were recorded 2 to 4 mm away at baseline (n 3) and during cromakalim (n 2). The difference between the cycle length of the reentry and the shortest captured interval was taken as the duration of the excitable gap. 8,12 Mapping-Electrode Plaque The plaque electrode array ( cm, consisting of 480 bipolar electrodes 1.6 mm apart) was described previously. 8,9,12,13 In 5 tissues, the plaque array was placed on the epicardial surface with a micromanipulator for action potential recordings after mapping. 9 Action Potential Recordings Transmembrane action potentials were recorded with glass capillary electrodes made from the most superficial epicardial cell layers. 9,10,14 Data were stored at 1 khz by use of Axotape 2.0 software (Axon Instruments, Inc). 9,10,14 Construction of Computerized Isochronal Activation Maps A custom-made, 509-channel computerized mapping system, EMAP (Uniservices) was used to construct isochronal activation maps. 8 10,12,15,16 Method of Dynamic Display of Activation Wave Fronts After manual editing of activation times, the pattern of activation was visualized dynamically. 8 10,12,16 An activated site first illuminates red, then yellow, then green, and then it fades away. Each illumination was selected to persist for 8 ms. Method of Measuring the Core of the Reentrant Wave Front During dynamic display, we traced the trajectory of the tip (ie, the innermost edge) of the reentry by advancing its motion 10 ms at a time until the front made a complete rotation. The perimeter traced by the tip trajectory defined the core of the reentry. 8,12,13,16 The computer then counted the number of 1.6-mm-apart electrodes encircled by the tip and calculated the core perimeter and its surface. 16 Cromakalim Superfusion At the end of control studies, the effects of 10 mol/l cromakalim (Beecham Pharmaceuticals) 2,4 on vulnerability to reentry by an S 2 (n 5), action potential, and reentry characteristics were tested 20 minutes after superfusion (n 6). Histological Analysis At the conclusion of the studies, all tissues were fixed in 10% formalin and stored in a refrigerator. 8,12 Five to 8 different transverse sections within the mapped regions were made and stained with hematoxylin and eosin. 12 Simulation Studies Computer simulation was performed in 2 2-cm tissues by use of the following continuous cable equation C m V t I LR 1 S v 1 R x 2 V x 2 1 R y 2 V y 2 where C m 1 F is membrane capacitance, S v 2000 cm 1 is the surface-to-volume ratio, R x 0.5 k cm is intracellular resistance in the longitudinal direction, R y k cm is intracellular resistance in the transverse direction (anisotropic ratio of 2.5), V (in mv) is the potential, and I LR is the total ionic current density from the phase I Luo-Rudy (LR) model. 17 The cable equation is integrated by an algorithm that uses operator splitting and time-adaptive integration methods developed by us (Z.Q. and A.G., unpublished data, 1998). The differential equations of the gating variables were integrated by the method developed by Rush and Lasen. 18 The remaining numerical integrations of the differential equations were done by the forward Euler method, with a space step equal to 0.02 cm and a variable time step ranging from 0.01 to 0.1 ms. The tissue was discretized into space units, with no-flux boundary conditions. We found that the following modifications of the LR model were necessary to make the induced spiral wave stationary to measure the core size. G Na was made equal to 16 ms/cm 2, Gsi 0, and the j gate was clamped to 1. The effects of different degrees of shortening of the APD on the spiral-wave period were evaluated by the progressive increase in the maximum conductance of the time-independent potassium channel (GK1) from 0.0 to ms/cm 2 (Table 2). A spiral wave was induced by cross-field stimulation, 5 and core size was measured after each increment of GK1, as described above (n 5). Statistical Analysis Student s t test was used to compare the APD, DET, ERP, S 1 -S 2 intervals, current strengths, mean cycle length, and core size before and after cromakalim. 8,10 Linear regression analysis was done to correlate the length of the linear core to the reentry cycle length, both in experiments and in simulations with StatView 4.5 and GB-STAT statistical software. 8,19 Results are expressed as mean SD. A value of P 0.05 was considered significant. Results Effects of Cromakalim on Epicardial Transmembrane Action Potentials Within 20 minutes, 10 mol/l cromakalim superfusion shortened the APD from a mean of to 48 7 ms (P , n 6). Shortening of the APD was associated with a significant (P 0.001) shortening of the ERP from to 45 8 ms(n 6) 3,4 and no change of the DET ( versus ma) (Table 1).

3 706 Acceleration of Reentry Figure 1. Sequential isochronal activation maps and 2 simultaneous action potential recordings from 2 cells (bottom recordings) in an isolated canine thin epicardial slice. A, Activation map during regular pacing (600 ms cycle length) before cromakalim. Numbers indicate electrode location and activation time. The start of the S 1 was taken arbitrarily as time zero. B, Map constructed 30 minutes after cromakalim (10 mol/l) superfusion. Bottom recordings are 2 action potentials recorded from sites indicated by single and double asterisks in A and B. Single-headed arrows in each panel indicate pacing site; double-headed arrow shows long axis of fiber orientation. Effects of Cromakalim on Conduction Figure 1 shows simultaneous action potential recordings from 2 epicardial cells before (A) and after (B) cromakalim. Conduction time between these 2 cells and the total tissue activation time were similar before and after cromakalim, which is compatible with previous results. 20 In all 7 tissues that we mapped, neither conduction slowing nor block was observed before or after cromakalim, which indicates the absence of drug effect and anatomic obstacles. The propagation, however, was somewhat nonuniform and anisotropic, with significantly (P 0.01) faster speed along (64 14 cm/s) than across (26 10 cm/s) the fiber (anisotropic ratio of 2.5). Cromakalim had no significant effect on conduction velocity during regular pacing (Figure 1) either along (63 17 cm/s) or across (24 13 cm/s) the fiber orientation (n 6). Cromakalim and Vulnerability to Reentry Induction The induction of reentry with an S 2 stimulus before and after cromakalim critically depended on the S 1 -S 2 coupling interval and on the strength of the S 2 current. No reentry could be induced outside these critical time-zone intervals regardless of the S 2 current strength (Table 1). The bounded nature (rectangular) of ventricular vulnerability to S 2 is consistent with our previously published results in canine 9 and swine 16 isolated and superfused epicardial thin ventricular slices. Cromakalim significantly (P 0.05) increased the mean product of the vulnerable period and the current strengths (area of the rectangle, in ma-ms units) from to Effects of Cromakalim on Induced Reentrant Wave-Front Characteristics Cromakalim accelerated the rate of the reentry, decreased its core size, and maintained its activity longer. Acceleration Cromakalim significantly (P 0.001) decreased the cycle length of induced reentry from to ms (27 episodes, 11 tissues). Figure 2 shows a dynamic display of an induced reentrant wave front before and after cromakalim. The wave front rotated along a line of functional conduction block that was parallel to the long axis of the fiber orientation during control and after cromakalim superfusion. Reentry remained stationary for several consecutive reentrant cycles, as evidenced by the fact that the tip trajectories of consecutive reentrant activation were superimposable. Figure 3 is an isochronal activation map of the same reentry episode shown in Figure 2. The longer central core (central site of functional block) during control compared with cromakalim is evident. The direction of the rotation clockwise (16 episodes) or

4 Uchida et al February 9, Figure 2. Consecutive color-coded snapshots of an induced stationary reentry during control (top) and during 10 mol/l cromakalim superfusion (bottom). Each dot represents an activated electrode site, which first becomes red, then yellow, then green, before fading away. Persistence of each color is 8 ms. White arrows indicate direction of wave-front rotation. Central line inscribes trajectory of the tip of the reentry that encircles a rectangular (elliptic) core. Note that the long axis of the central elliptic core is longer at baseline, and its period is longer (135 ms) than after cromakalim (90 ms). Numbers under each frame are moments snapshots were taken, with time zero chosen arbitrarily as being when the tip of the reentrant wave front was in the center. Vertical double-headed arrows indicate fiber orientation. counterclockwise (11 episodes) had no influence on the reentry cycle length either before or after cromakalim. Core Size A major difference between the reentry circuit at baseline compared with that after cromakalim was the size of the central elliptic core around which the front rotated. Although linear functional conduction block was present in both cases, the length of the line of functional conduction block was significantly longer during control (Figures 2 and 3A) than during cromakalim (Figures 2 and 3B). In both cases, rotation occurred in an elliptical pathway that roughly inscribed the perimeter of a rectangle with a width equal to 1 interelectrode distance or 1.6 mm (Figure 2). The mean length of the line of functional conduction block was significantly (P 0.01) longer during control ( mm) than after cromakalim ( mm). Cromakalim significantly decreased the core area, from to mm 2 (P 0.01). A significant (P 0.01) positive linear correlation (correlation coefficient of 0.89) was present between the length of the line of functional block (L, in mm) and the reentry cycle length (CL, in ms): CL (ms) L (mm). During reentry, cromakalim did not change conduction velocity around the core, measured as the ratio of core perimeter and reentry period ( versus cm/s; P NS). Facilitation and Reproducibility In 4 tissues, once reentry was induced by an S 2 stimulus, 10 consecutive trials of the same S 2 stimulus were tested for reproducibility to reinitiate reentry. For this purpose, after reentry was terminated, the same S 2 stimulus was tested for a total of 40 trials before and 40 trials after cromakalim. During control, reentry was induced in 11 (27%) of the 40 trials, whereas after cromakalim, reentry was induced in 36 (90%) of the 40 trials (P 0.001). In 2 tissues exposed to cromakalim, reentry was induced during regular pacing at a cycle length of 400 ms with twice diastolic current threshold, a phenomenon never observed at baseline. Duration During control, induced reentry had a short life span. It often terminated within 10 cycles after its induction, with a mean life span of seconds. In nearly all episodes, reentry terminated when the core of an otherwise stationary reentry drifted to the tissue border. Reentry was stationary in only 2 tissues and lasted for 8 and 12 minutes, respectively. In these 2 episodes, we estimated the excitable gap duration (see

5 708 Acceleration of Reentry Figure 3. Isochronal activation map during induced reentry before (A) and after (B) cromakalim. Same episode as shown in Figure 2. Reentry in a clockwise direction is shown (white arrows). Selected electrograms are from areas numbered with white background. Numbers in map indicate activation time, and lines are 10-ms isochrones. below). In the presence of cromakalim, reentry lasted significantly (P 0.001) longer in each tissue studied. The mean duration of reentry after cromakalim was minutes (range, 30 seconds to 32 minutes). Excitable Gap Figure 4 shows action potential recordings during the induction of reentry by an S 2 stimulus. A distinct phase 4 diastolic interval is present between 2 consecutive reentrant beats before and after cromakalim. This suggests the presence of an excitable gap. The introduction of premature stimuli showed early diastolic capture between 2 reentrant beats. Figure 5 illustrates early capture (34% of the reentry cycle) in 1 of the 2 long episodes of induced reentry at baseline and after cromakalim (54% of reentry cycle). A fully excitable gap was present during reentrant excitation before and after activation of the K-ATP channels. Reversal of the Effect of Cromakalim No sustained reentry could be induced after 60 to 90 minutes of superfusion with cromakalim-free Tyrode s solution (n 3) (Figure 6). Only short runs of reentry (3 to 8 cycles) could be induced within a narrow range of S 2 current strengths, as was the case at baseline (Figure 6). The APD and ERP lengthened from a mean of to ms and from 43 8 to ms, respectively (P 0.01; 9 sites tested in 3 tissues) (Figure 6). Histology Histological analysis of the hematoxylin-and-eosin stained tissue sections showed no signs of cellular necrosis or

6 Uchida et al February 9, Figure 4. Simultaneous transmembrane action potential and bipolar recordings during induction of reentry by a premature stimulus (S 2 ) during regular pacing at 600-ms cycle length. A, During baseline; B, 30 minutes after 10 mol/l cromakalim superfusion. In both cases, note the presence of a diastolic interval between 2 consecutive reentrant action potentials. autolysis throughout the entire thickness of the isolated slices. The lines of functional block occurred across the long axis of the myocardial fibers. Computer Simulation: Effects of APD Reduction on Spiral-Wave Dynamics Figure 7 shows an induced rotating spiral wave before (A) and after (B) reduction of the APD by increasing the GK1 from to ms/cm 2. Shortening of the APD consistently resulted in shortening of the spiral-wave period, with a concomitant reduction of the central core size around which the spiral rotated (Table 2). The core had an ellipsoid shape in accord with the anisotropic medium. 5 A progressive shortening of the APD was achieved by progressively increasing (7 values) GK1 (Table 2). Simple linear regression analysis between the length of the major axis of the ellipse (x axis) and the period (cycle length) of the spiral (y axis) showed a significant (P 0.001) correlation, with a 0.98 correlation coefficient: CL L, where CL is the spiral period in milliseconds and L is the length of the major axis of the ellipse in millimeters. Discussion Acceleration of induced functional reentry with cromakalim and maintenance of the reentry for a longer duration after activation of K-ATP channels constitute the major findings of the present study. Simulation replicated the experimental findings: acceleration of the reentry period was associated with a decrease in the core size. Figure 5. Excitable gap determination by application of electrical stimuli during sustained reentry. Top (control) and bottom (cromakalim) panels both show action potentials during an induced reentry. During control reentry (cycle length of 186 ms), the earliest captured beat occurs at a coupling interval of 125 ms, indicating the presence of an excitable gap (33% of reentry period). Reentry cycle length decreased to 98 ms (cromakalim), and the earliest captured beat occurred at a coupling interval of 63 ms, with an excitable gap equal to 35% of reentry period. Increased Vulnerability The effects of cromakalim on ventricular tissue are similar to the effects of acetylcholine on atrial tissue. 7,8,21 Whereas the wavelength varies along different segments of reentrant circuits, 22 the overall shortening of the wavelength increases the chances of incorporating a reentrant wave front in a given tissue mass, a phenomenon described as pharmacologic enlargement of tissue mass. 7 Because conduction velocity did not change after cromakalim, it is likely that in our isolated muscles exposed to cromakalim, the shorter wave-

7 710 Acceleration of Reentry Figure 6. Transmembrane action potential recordings during induction of reentry by an S 2 stimulus and reversibility of cromakalim effect. A, Induction of reentry at baseline with S 2 stimulus (arrow) that lasts for 7 to 8 beats. B, 25 minutes after 10 mol/l cromakalim superfusion; S 2 stimulus (arrow) induces sustained reentry at cycle length of 105 ms. C, Induction of reentry by S 2 stimulus, 90 minutes after washout. Reentry cannot be maintained. lengths led to greater vulnerability to reentry. 23 Increased tissue vulnerability to reentry induced by shortening of the wavelength has also been suggested to maintain reentry for a longer duration, 23 whereas increasing the wavelength had an opposite effect. 23,24 Chi et al 25 showed in in situ hearts that pinacidil, a K-ATP channel opener, increased ventricular vulnerability to fibrillation in a canine model of sudden cardiac death. Similar profibrillatory and increased vulnerability to reentry by K-ATP channel openers was also reported in rabbit hearts. 20 Mechanism of Acceleration of Reentry Acceleration of reentry was consistently associated with a decrease of the central core size, both in tissue and in simulation studies. The shorter path length needed for completion of a full rotation while the speed of the propagation remained unchanged led to reentry acceleration. We could not ascribe reentry acceleration to cromakalim-induced removal of incomplete recovery of excitability in the reentrant circuit 26 because there was a fully excitable gap when cromakalim was absent. However, because stimulated beats during reentry did not advance the return cycle, it is possible that a partially excitable gap might be present soon after repolarization. Double-wave reentry 27 as a mechanism of acceleration is refuted because acceleration occurred with a single- and not a double-arm reentry. Functional reentry in the ventricle is compatible with a spiral wave of excitation. 5,9,11,16,28 The spiral-wave theory predicts that a decrease in the APD or ERP causes the core size to shrink. 6 Simulation studies using a relatively realistic ventricular cell action potential model 17 reproduced the experimental findings. Although increasing GK1 is not a realistic representation of cromakalim effect, it nevertheless provided the desired shortening effect on the APD and systematic evaluation of its impact on spiral period and core size. Our simulation studies in an isotropic medium also showed a positive linear correlation between core size and spiral period with GK1 changes (data not shown). Faster rotation periods around smaller cores were observed in normal hearts 28 and in the epicardial border zone of healed myocardial infarcts. 29 In atrial tissues, acetylcholine-induced acceleration of functional reentry is also consistent with core-size reduction. 10 Although our findings are made essentially with regard to 2-dimensional spiral waves, the possibility of similar effects of APD on the 3-dimensional scroll wave is raised by our recent findings using procainamide. 30 We found that procainamide, while

8 Uchida et al February 9, Figure 7. Consecutive snapshots of rotating spiral wave before (A) and after (B) an increase in GK1. Colors represent membrane voltage: red is depolarization, followed by yellow, then green, then light blue, and finally dark blue (resting state), with 8-ms persistence for each color. Numbers above each frame represent time (in ms). Revolution time with GK (A) is 52 ms, and it is 37.2 ms with GK At the end of each panel, the core (arrow) size decreases after acceleration (B). Vertical double-headed arrows show long axis of fiber orientation. Anisotropic ratio was 2.5. increasing the refractory period, also increases the core size and prolongs the period of functional reentry in in situ hearts (scroll wave) during ventricular fibrillation. 30 Although our results show that ERP shortening accelerates the 2-dimensional spiral-wave period by core-size reduction, more work is needed to clarify this issue in 3-dimensional scroll waves. Maintenance of Reentry Termination of reentry often occurs when the core drifts to the edge of the tissue and dies out. 5,8,12,16 In the presence of cromakalim, reentry remained stationary, and the core did not drift toward the edge of the tissue. Core stationarity might explain the longer maintenance of the reentry. The mechanism of cromakalim-induced stationarity is unknown. It is possible that the drug might eliminate the excitability gradient, a driving force that promotes spiral core drift. 5 Study Limitations It could be argued that in the superfused model, the lack of adequate perfusion in cells 600 m thick 31 may cause progressive changes in the deeper cell layers, confounding the effects of cromakalim. However, the reversibility of the effects of cromakalim, both with respect to reentry vulnerability and epicardial cell action potential properties, suggests that superfusion with oxygenated Tyrode s solution over both surfaces of the thin epicardial slices kept the tissue viable. The use of the area of a rectangle (product of current times interval) may be argued to overestimate vulnerability. However, its substantial increase (40%) after cromakalim indicates that the method, despite its pitfalls, provides a useful index for quantitative comparison. 8,9 Our method of measuring the excitable gap clearly showed that functional reentry was not devoid of an excitable gap, because premature stimuli TABLE 2. Effects of GK1 on APD and Spiral-Wave Properties GK1, ms/cm 2 APD, ms Cycle Length, ms Long Axis, mm Short Axis, mm Long and short axis represent major and minor axes of the elliptic core of the spiral wave, respectively. Simulation results using the L-R cell model (see text).

9 712 Acceleration of Reentry could capture the tissue. It is, however, possible that both partially (early after repolarization) and fully (late in diastole) excitable gaps might be present in the functional reentrant circuit. Finally, testing our hypothesis required formation of spirals with stationary cores, a requirement that was met by modifying the original LR model. Although the maximum conduction velocity and resting properties did not change in the modified model, APD restitution and conduction velocity did. A recent study 32 emphasized the need for such changes for the induction of a stationary core. Clinical Significance The ability of cromakalim to accelerate and increase the upper limit of vulnerability for the induction of reentry might have clinical relevance. With the increased clinical use of potassium channel openers in the treatment of angina, hypertension, and asthma, their potential to accelerate reentry may convert reentrant ventricular tachycardia to ventricular fibrillation. 25,29 The clinical significance of these agents in increasing the upper limit of vulnerability remains to be elucidated. Acknowledgments This study was supported in part by a National Institutes of Health (NIH) SCOR grant (HL-52319), an NIH FIRST Award (HL-50259), the Cedars-Sinai ECHO Foundation, the Ralph M. Parsons Foundation (Los Angeles, Calif), an American Heart Association (AHA) National Center Grant-in-Aid ( N), an AHA Wyeth-Ayerst Established Investigatorship Award, and the UC Tobacco Related Disease Research Program (6RT-0020). We thank P.K. Shah, MD, for his support, Avile McCullen and Mei Ling Yuan for technical assistance, and Elaine Lebowitz for secretarial assistance. References 1. Wilde AA, Janse MJ. 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