Cellular Graded Responses and Ventricular Vulnerability to Reentry by a Premature Stimulus in Isolated Canine Ventricle
Background The cellular mechanism by which a point strong premature stimulus (S2) induces reentry is unknown. We hypothesized that cellular graded responses induced by an S2 mediate and control tissue vulnerability to reentry.
Methods and Results Reentry is induced in normal canine ventricular epicardial slices (30×38×2 mm, n=30) by an S2 at intervals shorter than the effective refractory period. The S1 is applied at the edge and the S2 at the center of the tissue. The line connecting the S1-S2 sites is parallel to the long axis of the fiber orientation. Isochronal activation maps were constructed with 56 to 480 bipolar electrodes, and the activation pattern was visualized dynamically. Reentry induced by an S2 is mediated by the graded responses as follows: The induced graded responses propagate with decrement toward recovered cells. When the amplitude of the propagated depolarizing graded responses reaches threshold relative to the recovering cells, an action potential is initiated along the fiber 2 to 3 mm away from the cathode of the S2. The distally initiated activation wave front blocks near the S2 site because the same S2-induced graded response prolongs the refractory period. The “broken” wave front then circulates around both sides of the block and reenters when the site of block recovers its excitability, completing the first figure-eight reentry cycle. Reentry cannot be induced when the S2 strength is >72±21 mA (upper limit of vulnerability) because these strong S2-induced graded responses convert the unidirectional block to bidirectional block by excess prolongation of the refractoriness.
Conclusions We conclude that the magnitude and the propagation of S2-induced cellular graded responses mediate and control vulnerability to reentry in the ventricular epicardium.
What is the average strength of the current necessary to produce paralysis of the heart (fibrillar tremulations of the ventricles) in man? By average current strength I mean a strength that is not too low, because in this case we do not obtain any deleterious effect, neither too high, because the heart is no longer paralyzed… .” Frederic Battelli, 18991 (translated from French).
The need for a critical current strength, one that is neither too high nor too low, to induce VF was recognized by Battelli1 before the turn of the century. Since then, it has been shown that the critical stimulus must be applied during a finite period of the cardiac cycle, the vulnerable period.2 3 4 5 6 The lower and upper levels of current strengths needed for VF induction, recently reconfirmed to be present in humans,7 were called the lower limit of vulnerability, or VF threshold, and the ULV, respectively.8 Using computerized mapping in the in situ ventricles, Chen et al9 showed that VF induced by a critical-point S2 stimulus was initiated by a figure-eight reentry around the S2 site. It was later shown that a shock applied across one side of the mapped region also induced VF. In the latter case, VF was initiated by a single-loop reentrant wave front when a specific voltage gradient field (5 V/cm) interacted with the recovering tissue, “the critical-point hypothesis.”10 Reentry would then occur around the area with a 5-V/cm voltage gradient.10 11 More recently, however, Chen et al12 proposed an alternative hypothesis for the mechanism of S2-induced reentry at the onset of VF in the intact in situ heart. According to this hypothesis (Fig 1A⇓ and 1B⇓), ie, the “propagated graded-response hypothesis,”12 reentry initiated at the onset of VF is caused by the ability of S2-induced graded responses first to prolong refractoriness near the S2 site and second to propagate away from the S2 site.13 The propagated graded responses encounter recovered cells and initiate activation at a site distant from the S2 site. The distally originated wave front propagates in all directions but blocks near the S2 site as a result of the prolongation of the refractoriness by the same S2-induced graded response. The wave front, however, propagates around both sides of the functional conduction block and reenters through the site of the initial block when it is no longer refractory, thus completing the first figure-eight reentry cycle (Fig 1A⇓ and 1B⇓). If this hypothesis is correct, then an S2-induced reentry leading to VF should arise from the interaction of propagated graded responses with the recovered cells distant from the S2 site. If the S2 strength is too low, the depolarizing graded response it induces may be too small to propagate toward recovering cells and/or too weak (subthreshold) to evoke a regenerative response at a distal site. Alternatively, with low currents, the prolongation of the refractory period near the S2 site may not be sufficient to result in unidirectional conduction block. Either mechanism may prevent reentry. Conversely, if the S2 strength is too high, bidirectional conduction block may occur because of increased prolongation of the refractoriness by the graded responses near the S2 (Fig 1B⇓). In this case, reentry is also prevented. The purpose of the present study was to use combined computerized mapping technique and cellular transmembrane potential recordings in isolated normal canine ventricular epicardium to test the propagated graded-response hypothesis of vulnerability to reentry by an S2.
Twenty adult mongrel dogs of either sex weighing between 15 and 18 kg were anesthetized with sodium pentobarbital (30 to 35 mg/kg IV). The hearts were then rapidly removed and placed in cooled oxygenated Tyrode’s solution. Thirty thin epicardial slices were isolated, 24 from the right and 6 from the left ventricles. The tissues were placed in a tissue bath (5×6×1 cm) and superfused with oxygenated Tyrode’s solution at 10 to 12 mL/min. Initially, we used 3×3-cm, 2-mm-thick tissues when we used 56 bipolar electrodes for mapping (n=16). In later experiments, when the number of channels of our mapping system was increased to 480 bipolar electrodes, we used 3.2×3.8-cm, 2-mm-thick blocks of tissue (n=14) to match the larger plaque size of the electrode array. For right ventricular epicardial tissues, the upper edge of the tissue block was always located near the base of the right ventricle, within 10 mm of the pulmonary valve. This specific site was chosen (1) because of the availability of extensive in situ activation mapping data9 12 and (2) because the graded-response hypothesis was tested at this specific site in the in situ ventricle.9 12 For the left ventricular preparations (six epicardial slices), the tissue block was isolated from the anterior surface adjacent to but not including the left anterior descending coronary artery. The S2-induced graded-response characteristics were similar in the right and the left ventricular tissues; therefore, the results were pooled together. In hearts from which two tissue samples were taken, the second trimmed tissue was kept in cold (4°C to 8°C) and continuously oxygenated Tyrode’s solution until use (about 2 to 2.5 hours). The viability of the second tissue sample was ascertained by the presence of a diastolic excitability threshold of <0.4 mA and of resting membrane potentials more negative than −75 mV of cells on the epicardial surface. All tissue samples in the bath were superfused with oxygenated Tyrode’s solution, and the temperature of the bath was maintained at 36.5±0.5°C and the pH at 7.4±0.1. The composition of the Tyrode’s solution (in mmol/L) was NaCl 125, KCl 4.5, NaH2PO4 1.8, NaHCO3 24, CaCl2 2.7, MgCl2 0.5, and dextrose 5.5 in triple-distilled, deionized water.14 15
The epicardial tissues were mounted in the tissue bath, and the fiber orientation was first determined grossly and later confirmed by histological analysis. One bipolar stimulating electrode was placed at the left edge of the tissue for regular (S1-S1) pacing at cycle lengths of 600 ms, and the second bipolar stimulating electrode was placed in the center of the tissue (Fig 1C⇑ and 1D⇑). With this arrangement, the line connecting the two stimulating electrodes was aligned along the long axis of the fiber orientation. The stimulating electrodes were made of silver wires that were Teflon coated except for their tips and had an ID of 0.1 mm and an interelectrode distance of 2 mm. The cathode side of the central stimulating electrode was located toward the S1 stimulating electrode and the anode side opposite the S1 site (Fig 1C⇑). The distance between the S1 and the S2 was about 1.5 cm. A Bloom stimulator (model OTU-110) was used to drive the tissues with constant current. The regular S1 electrical stimuli were of 2-ms duration with twice the diastolic threshold current.14 15 After nine regularly driven impulses, a premature stimulus (S2) of 5-ms duration was applied through the central stimulating electrode with current strengths of 1 to 100 mA. The S2 stimuli were applied at intervals 50 ms shorter than the ERP. The ERP near the S2 site was determined by the extrastimulus method and was defined as the longest S1-S2 coupling interval during which a stimulus of 5-ms duration and twice the diastolic threshold current does not initiate a propagated action potential.
Computerized Mapping of the Activation Patterns
The activation patterns were mapped with a custom-made computerized mapping system.16 17 18 Initially (n=16), we used 56 bipolar electrodes (mounted on a 3×3-cm electrode array) to construct activation maps. In the subsequent tissues (n=14), the activation maps were constructed with 480 bipolar electrodes that were mounted in a 3.2×3.8-cm electrode array. With the 56-electrode array (stainless steel wires, 0.1 mm in diameter), the distance between the poles of each bipole was 0.5 mm, and the interelectrode distance was 2 mm at the center (26 bipoles) and 5 mm at the periphery of the array (30 bipoles). In the 480-bipolar-electrode configuration, the distance between the poles of each bipole was 0.5 mm, and the interelectrode distance was 1.6 mm in the entire array. In the case of 56 electrodes, the electrode plaque was mounted on a manual micromanipulator (M3301, WPI) and slowly advanced until a gentle contact with the tissue was made. In the 480-electrode configuration, the mapping electrode plaque was mounted at the floor of the tissue bath, and the epicardial surfaces of the tissue slices were directly mounted on the plaque. Each electrode was made of insulated stainless steel wires with a diameter of 0.4 mm. The electrodes protruded 3 mm from the electrode mapping plaque to allow adequate superfusion of the epicardial surface with the Tyrode’s solution. The times of activation were determined by the computer according to our previously described algorithm.16 17 18 Manual editing was performed for each activation on each channel to ensure correct marking of the activation times by the computer. In seven tissue samples, the activation pattern after the S2 stimulus was visualized dynamically on the computer screen.17 18 Briefly, when an electrode site registers an activation, it initially becomes illuminated red, then yellow, then green, then light blue, and finally dark blue before it fades away. Each illumination was selected to persist for 10 ms, a duration that does not represent true recovery times.17 18 Selected color snapshots were obtained on a hard copy (Hewlett Packard Jet XL 300).
Transmembrane Action Potential Recordings
Two transmembrane action potentials were recorded simultaneously from the most superficial (first three) epicardial cell layers with machine-pulled glass-capillary electrodes with two differential amplifiers (Am-2 and ME-3221, Biodyne Electronics Laboratories)14 15 (Fig 1C⇑ and 1D⇑). On the basis of the results of initial activation map data, one recording was from the area of S2-induced conduction block and the second from the region of the earliest activation after the S2.
Characteristics of S2-Induced Graded Responses
We measured the amplitude, duration, takeoff potential, and rise time of the S2-induced graded responses. Fig 1E⇑ shows the method of measurements. The effects of the graded responses on the APD and the ERP were also determined. For this purpose, after the S2 stimulus, a stimulus of twice the diastolic threshold current was applied at the same site as the S2 at progressively shorter intervals until block. The longest interval that did not initiate an action potential was the ERP.
Propagation and Propagation Velocity of Graded Responses
To determine how far the graded responses propagate, a relatively large-amplitude graded response was first induced by the S2. Sequential microelectrode recordings were then made along and across the fiber at increasing distances (1-mm increments) from the cathodal and anodal sides of the S2. To calculate the conduction velocity of the graded responses, we induced graded responses near the S2 site that propagated to the distal cell and initiated an action potential. The difference between the rise time of the proximal-cell graded response and the upstroke of the distal-cell action potential is the conduction time between the two cells. Dividing the distance by the conduction time yields the conduction velocity of the graded responses.
Determination of Myocardial Fiber Orientation
At the conclusion of the electrophysiological studies, the locations of the S1 and the S2 stimulation sites were marked by pins, and all four edges of the tissue were stained with dyes of different colors. All tissue samples were then fixed in 10% neutral buffered formalin and stored for at least 24 hours at 4°C. The tissue samples were then sectioned 5 μm thick and stained with hematoxylin and eosin. The angle formed by the S1-S2 axis and the long axis of the epicardial fibers was then determined in each tissue sample by planimetry.
Comparison of the conduction velocity of the graded responses with the conduction velocity mediated by all-or-none action potentials was done by ANOVA and modified t test. Pearson correlations and linear regression analysis were performed between the ERP and the APD and between the amplitude and the duration of the graded response.
A value of P≤.05 was considered significant. All data are presented as mean±SD.
Characteristics of S2-Induced First Reentrant Wave Front
Fig 2⇓ shows a color-coded isochronal activation map of an S2-induced figure-eight reentry in an isolated right ventricular epicardial slice. Fig 2A⇓ shows the anisotropic propagation of the wave front during regular pacing (600-ms cycle length). The S1 pacing site is at the lower edge of the figure, and the fiber orientation is north-south. Conduction velocity was faster along the fiber than across the fiber (70 versus 52 cm/s). Fig 2B⇓ shows that an S2 applied in the center of the tissue initiates the earliest activation 3.2 mm away from the stimulus site toward the S1 site, with a line of apparent conduction block (thick black line in center). The apparent conduction block is functional because no block is present during regular pacing (Fig 2A⇓). The distally originating wave front breaks at the site of functional block into two wave fronts that continue to propagate around both sides of the line of block and reenters through the initial site of block (thick line “frame line,” Fig 2C⇓). The selected electrograms in Fig 2D⇓ show the sequential activation pattern (1 to 5) through the left arm of the figure eight. We induced similar figure-eight reentry by a critical S2 in all 12 tissues that we have mapped, consistent with previous findings in in situ ventricles.9 12 Fig 3⇓ is an enlarged map of Fig 2B⇓ and shows in greater detail the electrograms near the site of block and at the earliest site of activity. This consistent outcome after a vulnerable S2 guided us to target single cell recordings.
Vulnerability to Reentry
Time domain. Tissue vulnerability to reentry, systematically tested in six tissues, was critically dependent on the timing of the S2 stimulus. The S1-S2 coupling intervals that initiated reentry (“the vulnerable period”) were confined to the period that preceded the ERP by up to 50 ms (Fig 4⇓). The mean ERP at the S2 site during pacing at 600-ms cycle length was 168±15 ms. The mean shortest coupling interval that initiated reentry was 128±15 ms, and the longest was 155±20 ms, with a mean vulnerable period duration of 25±6 ms. Fig 4⇓ shows examples of strength-interval plots from three different tissues.
Current domain. Reentrant activity could be induced only when the current strength of the S2 was above and below a well-defined value. Current strengths falling outside this region could not induce reentry even if current was applied during the vulnerable period (Fig 4⇑). The mean threshold current strength for reentry induction (ie, the lower limit of vulnerability) was 28±13 mA, and the mean strongest S2 current above which no reentry could be induced (ie, the ULV) was 72±21 mA. These results are qualitatively compatible with earlier findings of reentry induction by a premature stimulus in isolated tissues,19 in in situ ventricles at the onset of VF,6 and in humans.20 The presence of a ULV is a unique feature of this model that allowed us to study the cellular basis of the ULV.
Relation Between S2 and Graded Responses
The properties of the graded responses, defined as a depolarizing response that manifests continuously variable configuration as a function of the S2 stimulating current, were evaluated near the cathodal and the anodal sides of the S2 stimulus.
Effects of Current Strength
An increase in the strength of the S2 current from 5 to 100 mA at a fixed interval (ERP−20 ms) caused a progressive increase in the amplitude (2±0.8 to 63±13 mV) and duration (5±2 to 87±23 ms) of the induced graded responses (n=6). Fig 5A⇓ illustrates one example. No graded responses could be induced during the entire phase 2 (plateau) of the action potential no matter how strong the S2 was (see Fig 7B⇓ below). These findings are compatible with those of Kao and Hoffman21 seen on subendocardial fibers.
Effects of Coupling Interval
Similarly, an increase in the coupling interval of the S2 at a fixed current strength caused a progressive increase in the amplitude and the duration of the graded responses (Fig 5B⇑). The mean shortest and longest coupling intervals during which graded responses could be induced at a pacing cycle length of 600 ms were 115±8 and 170±12 ms, respectively (n=12).
Graded Response and ERP Prolongation
Fig 5C⇑ shows the relationship between the total APD and the ERP. In six tissue samples, a regression analysis of 78 different measurements showed a significant positive correlation (P<.01, r=.96) between the ERP and the total APD over 100 ms of APD prolongation: ERP (ms)=1.067×APD (ms)−24.51 ms.
Graded-Response Amplitude-Duration Relationship
A regression analysis of 187 induced graded responses in eight tissue samples showed a significant (P<.01, r=.79) positive linear correlation between the graded-response amplitude and the graded-response duration: Duration (ms)=0.98 ms/mV×amplitude (mV)+8.41 ms.
Voltage Dependency of Graded Responses
The graded-response amplitude was voltage dependent (Fig 5D⇑). As the takeoff potential became more negative (ie, −15 to −65 mV), the amplitude of the graded response induced by a given S2 current strength grew progressively. Similarly, for a given takeoff potential, the amplitude of the graded responses increased with increasing S2 current strength, consistent with the results of Knisley et al.22 Fig 5D⇑, a graph of pooled data from two tissues, shows the entire voltage range within which graded responses could be induced.
Anisotropic Propagation of Graded Responses
The induced graded responses propagated in a decremental and anisotropic fashion. Fig 6⇓ shows graded-response amplitude at increasing distances from the cathode of the S2 stimulus along and across the fiber orientation (pooled data from 10 tissues).
Graded Responses Near the Anodal Pole
Graded responses were also observed near the anodal pole of the S2 stimulus with characteristics that depended on the S2 stimulus parameters (Fig 7A⇓ through 7C). No graded responses could be induced at distances >3 mm for the anode or during the entire plateau range of the action potential for current strengths up to 100 mA (Fig 7A⇓). S2 applied at a slightly later part of the plateau induced small-amplitude (2- to 4-mV) graded responses, with a net shortening of the total APD (Fig 7B⇓). However, with relatively late-coupled S2 stimuli (ie, >110 ms), a graded response with a net prolongation of the total APD occurs (Fig 7C⇓), consistent with previous studies on Purkinje fibers23 and on endocardial ventricular muscle cells.24 At equal distances from the two poles of the S2 along the fiber (analysis done 2 mm away from each pole and with an S2 of 80 mA), the amplitude and the duration of the graded responses were significantly (P<.01) lower in the cells at the anodal side than in the cells at the cathodal side (6±2 versus 28±9 mV and 16±4 versus 36±10 ms, respectively).
Propagation of Graded Responses and Initiation of Action Potentials
When the amplitude of the propagated graded responses in the recovering cells near the S1 site reached threshold, an action potential was initiated. Threshold potential could be reached by the propagated graded responses either by increasing the coupling intervals of the S1-S2 or by increasing the S2 current strength.
Effects of the S2 coupling interval. Fig 8A⇓ shows an example of simultaneous recordings of action potentials from two cells (Fig 1C⇑). As the coupling intervals increased from 164 to 168 ms (Fig 8A⇓, toward the right), the amplitude of the graded responses increased from 25 to 38 mV in cell 1 and from 3 to 8 mV in cell 2. At a 170-ms interval, the propagated graded-response amplitude in the distal cell 2 reaches threshold and initiates an action potential (single arrow pointing upward). The 16-ms delay in the timing of the voltage rises between the two cells 2 mm apart corresponds to a conduction velocity of 12.5 cm/s. This velocity represents the velocity of the graded-response propagation. The smooth transition of the membrane potential from the phase 4 to the phase 0 upstroke of the action potential in the cells 2 strongly suggests that the action potential is initiated from the peak of the graded response in cell 2 (single upward arrow). The cell 2 action potential, however, blocks at cell 1 and causes an electrotonic depolarization in this cell (Fig 8C⇓). Electrotonically mediated depolarizations of similar magnitudes and voltage ranges were described by Cranefield et al25 in the ventricular endocardium.
Effects of the S2 current strength. Fig 8B⇑ and 8C⇑ illustrates the attainment of threshold potential in the distal cell 2 by the propagated graded responses with increasing S2 current strength and initiation of an action potential in this distal cell (upward arrow in Fig 8C⇑). The 13-ms delays in the timing of the voltage increases between the two cells 2 mm apart correspond to a conduction velocity of 25 cm/s. This velocity represents the velocity of the graded-response propagation. The distally originated action potential exerts an electrotonic depolarizing influence on the proximal cell as in Fig 8A⇑.
Comparative Conduction Velocities of Graded and Regenerative Responses
The conduction velocity of the graded responses was significantly (P<.001) slower than the velocity of the regenerative responses (18.2±3.8 versus 60±10.9 cm/s, respectively, n=6) (Figs 9⇓ and 10⇓). Propagation supported by combined graded and active responses was slower than the velocity supported by regenerative responses. Fig 10⇓ shows two examples in which the distal cells were located 10 and 11 mm away from the cathode, that is, outside the graded-response spread zone (see Fig 6⇑). In these cases, the initial 6 to 7 mm of the spread is mediated by the graded responses, whereas the final 4 to 5 mm is mediated by the regenerative responses. The mean combined velocity (n=6) was significantly (P<.001) slower (24.1±4 cm/s) than the velocity of all-or-none responses (64±10.9 cm/s) but faster (P<.024) than the velocity mediated exclusively by the graded responses.
Graded Responses and Initiation of Reentry
In five tissues, after an initial activation map, we recorded with one microelectrode from the area of the earliest activity and with a second from the area of local conduction block. Fig 11⇓ shows one such example. Fig 11A⇓ shows activation during regular pacing. Panel B shows that an S2 of 35 mA strength applied with a coupling interval of 150 ms caused a local block and distal early activation (two curved arrows) leading to the first reentrant wave front (panel C), as in Fig 2⇑. Subsequent simultaneous recordings of two transmembrane potentials (site of block and site of earliest activation, two dots in panels A through C) are shown in panels D and E. The distally originated front (panel B) rotates around the site of block, then reenters (panel C), initiating the first action potential (1 in panel E). This scenario is similar to the one shown in Fig 10C⇑.
Superstrong S2 Stimulus Prevents Reentry
Fig 12⇓ illustrates one example in which the ERP was progressively increased by the graded responses with progressive increases in the strength of the S2 current at a fixed interval. Increasing the current strength from 40 mA (panel A) to 50 mA (panel B) caused a graded response–mediated origination of action potential in the distal cell 2, which rotates around the site of block and reenters through this site of block after it recovers its excitability (panel B). In this case, the graded-response duration was 58 ms and the ERP 238 ms. However, when the current strength was raised to 80 mA (panel C), the graded-response duration became 102 ms and the ERP 288 ms. In this case, the distally originated action potential could not reenter. The excess increase of the ERP converts the unidirectional block (panel B) to bidirectional block and prevents reentry (panel C). An activation map with 480 electrodes showed that currents above the ULV induce bidirectional conduction block near the S2 site that prevents reentry. Fig 13⇓ illustrates one such example using dynamic display. The S2-induced distally originated wave front (Fig 13A⇓) propagates toward the S2 and blocks at this site (frame C, double white lines). The wave front then rotates around this site of block and reaches the opposite side (frame D, double white lines). However, once the wave front reaches the other side of the block, it undergoes another block (bidirectional block), and reentry is prevented (frames E through H).
Myocardial Fiber Orientation
Histology showed that the S1-S2 electrical axis of stimulation was always along the fiber orientation (roughly parallel to the long axis of the fiber). The angle formed by the two axes (the S1-S2 electrical axis and the long axis of the fibers) ranged between 0° and 30°, with a mean±SD of 8.8±10.1°.
Graded Responses and Reentry
The major finding of this study is the ability of the propagated graded responses to initiate the first beat of reentry in a tissue that lacks significant initial nonuniformity of repolarization.26 We have shown that wave break27 leading to the first beat of reentry in an otherwise uniformly anisotropic tissue is caused by the spread of S2-induced graded responses that originate an activation at some distance from the S2 site and by the unidirectional block at the S2 site created by the same S2 stimulus. Fig 1⇑ shows schematically the events that lead to the initiation of the first figure-eight reentrant wave front induced by propagated graded responses.
Evidence for Propagation of Graded Responses
That the propagating graded responses and not the direct S2 stimulus field effect initiate a distal action potential is supported by the following observation. Activation of the distal cells occurs 20 to 60 ms after the end of the S2 stimulus (Figs 8 through 11⇑⇑⇑⇑). With direct field effects, no such long delays would occur. Furthermore, when these same strong S2 stimuli are applied after full recovery so as to initiate active regenerative responses, the delay in the distal cell activation shortens.
These findings show not only that the distal cell is activated by the proximal cell response but also that the velocity of the all-or-none responses is faster (3 to 5 times) than the conduction velocity of the graded responses over the same path. The possibility of an alternative conducting path between the proximal and the distal cell is unlikely, because the bipolar electrode located in the middle of the two cells remains inactive during the entire period of the delay. The demonstration of propagating graded responses in the epicardium in the present study is consistent with the results of van Dam et al13 and Ino et al,28 who showed similar phenomena in the canine ventricular endocardium. Furthermore, the demonstration of epicardial “phase 2” reentry29 30 induced by the propagation of the dome (secondary slowly rising depolarization) of the action potential is compatible with the propagation of the graded responses.
Graded Response and Critical-Point Hypotheses of Reentry
Although both hypotheses attempt to provide a mechanism for reentry and implicate graded response–induced anisotropic prolongation of the ERP in reentry formation,10 22 there are nevertheless a number of important differences between the two. (1) In the critical-point hypothesis, developed in Dr Ideker’s laboratory, the S2 that initiates reentry directly excites about 40% of the mapped tissue.10 No region of direct excitation exists in the graded-response hypothesis. (2) In the graded-response hypothesis, the region of block is the region of the graded-response distribution, ie, the region of graded response–induced prolongation of the ERP. In the critical-point hypothesis, no clear cellular electrophysiological descriptors are provided to explain the mechanism of the functional block. Rather, it is proposed that block occurs “when a line corresponding to one of the excitatory states intersects a line corresponding to one of the critical recovery states, (the critical point) … excitation wave front occurs on one side of the critical point [block] and then pivots around the critical point… .”22 This proposal is based on the postulate “that critical states of each of the excitatory and recovery processes occur when a stimulus of an appropriate strength is given at an appropriate time during the recovery.”22 (3) In the graded-response hypothesis, reentry occurs through the site of the initial block created by the graded responses near the S2. In the case of the critical-point hypothesis, reentry occurs through the site of direct excitation when this site recovers its excitability. (4) In the graded-response hypothesis, the first reentry cycle is in the form of a figure eight, whereas in the critical-point hypothesis, it is a single-loop reentry.10 11
The ionic mechanism that underlies the graded responses remains undefined at present. It is conceivable that both passive and active ionic mechanisms may participate in the depolarization process after a strong premature stimulus.13 Three properties of the graded responses argue against an exclusively passive mechanism: first, the inability of strong currents (anodal or cathodal) to induce a graded response at positive (plateau phase) transmembrane voltages (Fig 7⇑); second, the voltage dependence of the graded-response amplitude, ie, increase in amplitude with increasing voltage negativity, consistent with the report of Knisley et al22 ; and third, the incompatibility of the pattern of voltage decay of the graded-response amplitude along the fibers with the measured space constants (0.9 to 1.2 mm) in ventricular muscle fibers.31 32 33 Graded responses with amplitudes comparable to those found near the S2 source could be recorded at distances 2 to 3 mm from the source (Fig 6⇑). Such high-amplitude depolarization two to three space constants away from the source argues against exclusively passive ionic mechanisms.31 32 33 High-amplitude graded responses with overshoots were also observed by Kao and Hoffman21 in canine subendocardial cells and by Knisley and Hill11 on canine ventricular epicardium with optical recordings. Although the nature of the active currents involved in the initiation of depolarizing graded response remains undefined, it is possible that some sodium channels and/or calcium channels may be “forced” to be reactivated by the strong S2 depolarizing currents.
Our inability to induce graded responses during the plateau phase of the action potential appears at variance with the results of Dillon,34 who by using optical action potentials and field stimulation observed “plateau” effects in the rabbit heart. Optical action potentials, unlike single-cell transmembrane potential recordings, are “like monophasic action potentials” and therefore represent the spatial average of the underlying multicellular activity.35 Therefore, the observed optical “plateau effect” during field stimulation may be a reflection of spatial averaging effect and not a true single-cell effect. It is therefore possible that the different recording and stimulation approaches could at least partly explain the observed differences between our study and Dillon’s.34
Implications of Graded-Response Hypothesis
The graded-response hypothesis provides an explanation as to why the earliest site of activation after a critical S2 occurs between the S1 and the S2 sites along the fiber. The earliest site of activation is located along the fiber, because the graded responses propagate to a much greater extent and with higher amplitudes along the fiber and toward the cathodal side rather than across the fiber and toward the anodal side. Since cells near the S1 site depolarize first and recover first, they become the first target of early excitation by the propagating graded responses. Furthermore, the graded-response hypothesis can also explain the phenomenon of the ULV.6 7 8 Superstrong S2 stimuli, by virtue of the longer-duration graded responses they induce, cause excessive prolongation of the refractory periods (Fig 5⇑) that prevents timely recovery of the cells at the site of the initial block. The excessive prolongation of the refractory period converts the area of unidirectional block to bidirectional block, preventing reentry. It is conceivable that superstrong S2 stimuli, in addition to initiating activation along the fiber, may also initiate activation at other sites. Multiple sites of early activations may arise because superstrong S2 stimuli cause higher-amplitude graded responses that propagate across the fibers as well and might thus encounter recovered cells and initiate early activity (Fig 13C⇑).
Limitations of the Study
It may be argued that the absence of simultaneous activation map and action potential data may prevent accurate testing of the graded-response hypothesis of vulnerability to reentry by an S2. Although we used the sequential recordings approach, the activation map data showed that the earliest activation site and the site of functional block after an S2-induced reentry consistently occurred at predictable sites relative to the S1 and the S2 sites. Consistent locations of the earliest site and the site of functional block have also been documented in in vivo studies using similar stimulation protocols.12 In one tissue, repeat multisite mapping using identical premature stimulus parameters after the conclusion of the microelectrode recordings verified that both the site of the block and the site of the earliest activation were stable and occurred at the predicted sites. We therefore think that the method of sequential multisite extracellular studies followed by intracellular recordings provided the opportunity to determine the cellular mechanism of unidirectional block and early origination of action potential away from the S2 site.
The presence of similar reentrant wave fronts at the very onset of an S2-induced VF in the in situ hearts suggests that the graded responses may also be involved in reentry formation at the onset of VF.9 12 This contention gains support from the similarities of the (1) S2 stimulus and electrode characteristics, (2) reentrant wave front properties (figure eight), and (3) tissue anatomic location relative to the S1-S2 stimulation in the two in situ and in vitro models. In addition, the presence of a ULV for reentry in our in vitro model mimics the presence of a ULV in in situ canine hearts6 8 and in humans.7 Because the values of the ULV and the defibrillation energy requirements are closely related,7 the graded-response mechanism of ventricular vulnerability to reentry may also have relevance to the mechanism of ventricular defibrillation.
Selected Abbreviations and Acronyms
|APD||=||action potential duration|
|ERP||=||effective refractory period|
|ULV||=||upper limit of vulnerability|
This study was supported in part by an NIH Specialized Center of Research (SCOR) Grant for Sudden Death (HL-52319) and a FIRST Award (HL-50259) from the NIH; the Electrocardiographic Heartbeat Organization (Dr Karagueuzian); the Ralph M. Parsons Foundation, Los Angeles, Calif; and an American Heart Association (AHA) National Center Grant-in-Aid (92009820) and an AHA Wyeth-Ayerst Established Investigatorship Award (Dr Chen). We thank Drs Prediman K. Shah and James Forrester for their support, Dr Takanori Ikeda for helpful comments, Dustan M. Hough for HeartWatch software development, Avile McCullen and Meiling Yuan for their technical assistance, and Elaine Lebowitz for her secretarial assistance.
- Received June 27, 1996.
- Revision received November 19, 1996.
- Accepted November 25, 1996.
- Copyright © 1997 by American Heart Association
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