Measurements of Intramural Shock Induced A Vm in Wedge Preparations

Experiments in cell cultures provided information about the effects of shocks on Vm at the microscopic level. However, cell cultures are structurally and electrophysiologically different from the intact myocardium. Therefore, results obtained in cell cultures cannot be directly applied to the whole myocardium, which necessitates measurements of the shock effects in the whole tissue. Since no experimental methods to measure intramural Vm in the intact heart are currently available, an approach based on optical mapping of Vm on the cut transmural surface in isolated wedge preparations of left ventricular was used. Such a preparation consists of a portion of left ventricular (LV) wall maintained viable via perfusion through a branch of coronary artery. Uniform-field shocks were applied parallel to the cut surface in order to avoid formation of AVm due to the boundary conditions on this surface. Several optical mapping studies were carried out in these preparations to characterize the effects of shocks on transmural Vm.

In the first study,39 the whole transmural surface was mapped at a spatial resolution of ~1.2mm per diode. Shocks of variable strength (2-50 V/cm-1) were applied in the early phase of AP. It was found that effects of shocks on transmural Vm strongly depended on the shock strength. Relatively weak shocks (~2V/cm-1) induced positive and negative polarizations at the tissue edges facing the cathode and the anode, respectively (Fig. 9a). Changing the shock polarity reversed the polarization pattern. For shocks of both polarities, maximal AV+ and AVm were achieved at the wall edges, and there was a relatively gradual transition of AVm magnitude between the edges with relatively small (< 6% APA) local Vm changes in the wall middle. Shocks caused prolongation of action potential duration (APD)50 at sites of maximal AV+, whereas at sites of maximal AVm the APD50 was either not changed or slightly prolonged.

Increasing shock strength above ~4V/cm-1 produced several important changes in polarization patterns. First, such shocks produced localized positive and negative AVm inside the wall (Fig. 9b). Such isolated polarizations, as well as the overall nonuniform distribution of AVm across the wall, indicated the presence of intramural virtual electrodes. Second, shock-induced polarizations became strongly asymmetric, with negative AVm exceeding positive AVm measured at the same sites at the opposite shock polarity. This is similar to the AVm behavior observed in cell cultures. Third, negative AVm extended toward the cathodal side of the preparation. This was different from measurements in cell cultures where only positive polarizations were observed at the cathodal edge of cell strands. Fourth, shocks prolonged APD everywhere across the wall, including sites with both positive and negative AVm and AVm. This finding was also surprising because APD was expected to decrease at the sites of negative AVm where positive charges are removed from the intracellular space.

Increasing the shock strength further caused even more drastic changes in polarization patterns. Shocks with a strength of ~20V/cm-1 and larger of both polarities produced predominantly negative AVm across the whole transmural wall (Fig. 9c). In addition, shocks prolonged APD everywhere in the wall. The degree of APD prolongation was

Figure 9: Effects of shocks on intramural Vm in left ventricular wedge preparations. (a) Isopotential map of AVm (upper-panel ) and selected optical recordings of Vm changes (lower panel) induced by weak shocks (£~2V/cm-1). Left inset shows outlines of wedge preparation, shock electrodes, and mapping area. (b) AVm induced by shocks intermediate strength (E~9V/cm-1). (c) AVm induced by shocks high strength (E^28 V/cm-1) (from Ref. 39)

Figure 9: Effects of shocks on intramural Vm in left ventricular wedge preparations. (a) Isopotential map of AVm (upper-panel ) and selected optical recordings of Vm changes (lower panel) induced by weak shocks (£~2V/cm-1). Left inset shows outlines of wedge preparation, shock electrodes, and mapping area. (b) AVm induced by shocks intermediate strength (E~9V/cm-1). (c) AVm induced by shocks high strength (E^28 V/cm-1) (from Ref. 39)

i o n to similar for shocks of both polarities, and it was not dependent on the local AVm value. Again, these findings are at odds with observations in cell cultures as well as with basic biophysical principles postulating that (1) shocks should induce both positive and negative polarizations, reflecting inflow of shock current into intracellular space at some locations and outflow at other locations and (2) shocks should shorten APD at sites of negative AVm where charges are removed from the cell interior.

A limitation of the wedge preparation is that boundary conditions at the cut transmural surface are different from those in the intact myocardium. Since boundary conditions play a critical role in shock-induced AVm, it may be asked whether or not intramural polarizations are an artifact of the boundary conditions. To prove that this is not the case, intramural polarizations have to be demonstrated in the intact myocardium. Experiments in the wedge preparations showed that negatively biased intramural polarizations induced by strong shocks may extend to the wall surface facing the cathode electrode. It is well known that optical measurements from a surface reflect Vm changes spatially averaged over a certain tissue depth.55'56 Therefore, it is hypothesized that negative AVm could be measured on the epicardial surface when this surface is facing the cathode. Because only positive polarizations can be produced on the cathodal wall surface, registration of negative AVm on this surface would unequivocally prove the existence of intramural virtual electrodes.

This hypothesis was tested by measuring AVm on the intact epicardial surface in LV preparations stained with a Vm-sensitive dye using two methods: (1) staining via surface dye application (surface staining), and (2) staining via coronary perfusion (global staining).57'58 With the first method, a surface tissue layer with a thickness of approximately 0.25 mm was stained. In the second case, tissue was stained uniformly across the whole LV wall. Shocks (2-50V/cm—1) were applied in the epicardial-to-endocardial direction via transparent mesh electrodes. Shock-induced AVm were mapped through the epicardial electrode from the same locations after both surface and global staining. Optical recordings revealed significant differences between AVm measured in two staining conditions, and these differences were especially prominent for cathodal shocks (Fig. 10). Relatively weak cathodal shocks produced positive AVm in both staining conditions. However, AVm measured in the surface-stained tissue were much larger than those measured in the globally stained tissue (Fig. 10c, d). At higher shock strength, cathodal AVm measured in globally stained tissue became uniformly negative, whereas in surface-stained tissue they remained positive (Fig. 10a, b, black traces). These differences in the magnitude and polarity of AVm induced by cathodal shocks in surface- and globally stained tissue can be explained by the presence of intramural virtual electrodes in the subepicardial tissue layers.

The most important finding from these experiments is that shocks cause widespread polarizations in intramural myocardium. The mechanism of these polarizations, however, remains uncertain. It is unlikely that they were due to nonuniform shock field because the electrical field in the bath was uniform without preparations. Therefore, it is more likely that intramural AVm were due to nonuniform tissue structure. It is also likely that two different types of AVm were due to different structural properties. The isolated areas of positive or negative AVm induced by shocks of moderate strength were probably caused by relatively large-scale nonuniformities such as fiber rotation, variation in the intracellular

Figure 10: Detection of subepicardial intramural AVm in the intact tissue. (a) Optical recordings of epicardial Vm during application of cathodal (black trace) and anodal (gray trace) shocks with E « 14V/cm—1 in a surface-stained preparation. Measurements were performed through an opening in epicardial mesh electrode. (b) Corresponding Vm recordings in a globally stained preparation. (c, d) Dependences of corresponding averaged AVm magnitudes on the shock strength. Curves are second order polynomial fits of data (from Ref. 58)

Figure 10: Detection of subepicardial intramural AVm in the intact tissue. (a) Optical recordings of epicardial Vm during application of cathodal (black trace) and anodal (gray trace) shocks with E « 14V/cm—1 in a surface-stained preparation. Measurements were performed through an opening in epicardial mesh electrode. (b) Corresponding Vm recordings in a globally stained preparation. (c, d) Dependences of corresponding averaged AVm magnitudes on the shock strength. Curves are second order polynomial fits of data (from Ref. 58)

volume fraction, or blood vessels. As expected from AVm produced by large nonuniformities, they changed their sign with a change in the shock polarity.

Intramural polarizations of the second type, which remained negative for shocks of both polarities, are likely to have a different anatomic substrate. It is hypothesized that these AVm are due to microscopic discontinuities in the tissue structure associated with collagen septa that are present in the LV wall at a high density.39 Such layers have microscopic thickness. Therefore, their Vm response to electrical shocks should be similar to the behavior of microscopic cultured cell strands. Particularly, shocks are expected to induce both positive and negative AVm on the opposite sides of cell layers. However, because these polarizations were measured on a macroscopic scale (1.2 mm), negative and positive polarizations should be averaged out. When cardiac tissue has a linear Vm response to electrical field, the net result should be zero or a negligible macroscopic polarization. This explains the absence of intramural AVm during weak shocks when Vm response is linear. Stronger shocks, however, induce nonlinear AVm with a strong negative bias (AVm > AV+ during AP plateau. Because of this asymmetry, macroscopic measurements should yield only negative AVm. This can potentially explain globally negative polarizations observed in wedge preparations.

The logical test of the hypothesis postulating the existence of microscopic polarizations is mapping of AVm at a high spatial resolution. Therefore, transmural AVm was mapped at a tenfold higher optical magnification (0.11 mm/diode-1 vs. 1.2mm/diode-1).59 As shown previously, in low-magnification recordings AVm produced by strong shocks were globally negative, extending to the wall edge for both anodal and cathodal shocks (Fig. 11a, b). In contrast, high-magnification recordings at the wall edge revealed positive AVm for cathodal shocks (Fig. 11d) and negative AVm for anodal shocks (Fig. 11c) for all shock strengths. Positive A Vm were also observed at high magnification in the middle of the wall (not shown). However, alternation of positive and negative AVm, expected from microscopic secondary sources, was not found. This can be explained by the fact that optical resolution does not scale up with increasing optical magnification. Indeed, it was shown in a mathematical model of light propagation that, due to light scattering in three-dimensional cardiac tissue, an increase in optical magnification leads only to a modest increase in resolution.55 Even when the size of the imaged area becomes negligible, dimensions of the interrogated tissue volume remain relatively above several hundred microns.

The excitatory hypothesis of defibrillation mechanism postulates that shocks cause direct and simultaneous activation of the majority of excitable or partially excitable tissue. To test this hypothesis, transmural activation patterns induced by shocks applied during diastolic phase of cardiac cycle in wedge preparations were measured.40 It was found that during the weakest shocks (~1-4V/cm-1) applied in diastole, earliest activation occurred predominantly (but not exclusively) on the cathodal side of preparations. The time of transmural spread (several milliseconds) was significantly shorter than the activation time after local epicardial stimulation, indicating that transmural activation was the result of the direct tissue activation by a shock of some areas as well as of impulse propagation from these directly excited areas. During shocks of intermediate strength (^4-23 V/cm-1), activation was initiated at multiple transmural sites from where it rapidly (within ¡1ms) spread across the whole LV wall. Very strong shocks (^23-44 V/cm-1) could cause discontinuous activation, where some areas were activated immediately on the shock onset and other areas were activated with a large delay. In all cases, the sites of the earliest activation corresponded to the areas of largest AVm measured during AP plateau; the sites of delayed activation observed during the strongest shocks corresponded to the areas of minimal plateau AVm. Thus, diastolic shocks with a strength varying over a wide range cause direct and nearly simultaneous activation of the whole LV wall. Sites of earliest and latest activation correspond to areas of maximal and minimal AVm measured during shocks applied in AP plateau. These findings support the excitatory hypothesis of defibrillation.

Figure 11: The role of optical magnification in measurements of intramural AVm. (a, b)Low-magnification (0.85x ) measurements of AVm in the subepicardial transmural region of LV wall during action potential plateau. Resolution equals 1.2mm/diode-1. Shock strength E « 21 V/cm-1. (c, d) High-magnification (10x) measurements of AVm from the area shown in (a) and (b) by the thick rectangle. Thin rectangles correspond to individual photodiodes. Thick lines in maps depict boundaries between areas of positive and negative AVm. Black and gray traces display plateau AVm inside and outside of the high-magnification mapping area (from Ref. 59)

Figure 11: The role of optical magnification in measurements of intramural AVm. (a, b)Low-magnification (0.85x ) measurements of AVm in the subepicardial transmural region of LV wall during action potential plateau. Resolution equals 1.2mm/diode-1. Shock strength E « 21 V/cm-1. (c, d) High-magnification (10x) measurements of AVm from the area shown in (a) and (b) by the thick rectangle. Thin rectangles correspond to individual photodiodes. Thick lines in maps depict boundaries between areas of positive and negative AVm. Black and gray traces display plateau AVm inside and outside of the high-magnification mapping area (from Ref. 59)

They also indicate that shock-induced activation is caused by formation of microscopic intramural secondary sources.

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