The Role of Microscopic Tissue Structure in the Shock Effects Experiments in Cell Cultures

Until recently, the effects of microscopic tissue structure on defibrillation were investigated almost exclusively in computer models.12-15'21 Experimental studies of structural effects in the heart are hampered by the inability to measure Vm at the microscopic level and by the three-dimensional complexity of cardiac muscle that prevents precise correlation of Vm changes with the tissue structure. Also, because cardiac muscle contains structural discontinuities of multiple types, separating the effects of one individual structure from another as well as from effects of other structure-independent factors is extremely difficult. These obstacles can be overcome using cultures of cardiac cells. The main advantage of cell cultures is that they grow as two-dimensional monolayers that allow precise microscopic measurements of both the tissue structure and electrophysiological parameters and their correlation. In addition, the monolayer structure can be modified in a desired way using techniques for patterned cell growth, which greatly facilitates structure-function studies. Electrophysiological parameters of cell cultures such as the maximal upstroke rate of rise and the conduction velocity are quite similar to those measured in adult ventricular tissue.22

The use of cell cultures allows Vm measurements at microscopic resolution using the optical mapping technique. This method involves staining of tissue with a voltage-sensitive dye and measurement of either dye absorption or, more often, dye fluorescence, using an array of photodetectors. This method has been widely used for multisite recordings of Vm from brain and cardiac tissue. It has several important advantages over conventional recordings that use electrodes. One of the main advantages is that optical mapping allows simultaneous measurements of Vm at hundreds or thousands of locations, whereas electrical Vm recordings are limited to just a few sites. Another advantage is that optical signals are devoid of stimulation artifacts, which is especially important in defibrillation studies where strong artifacts created by defibrillation shocks interfere with electrical measurements of Vm during shocks and 20-50 ms after the shocks. A disadvantage of optical mapping is that the optical signals reflect only relative changes of membrane potential. The absolute value of optical signals depends on multiple factors, including the density of dye staining, degree of dye internalization, uniformity of excitation light intensity, and others. Combination of these factors results in a significant variability of fluorescence intensity throughout a preparation, independently of the underlying variation of Vm. The Vm-independent variability of optical signals can be somewhat reduced by measuring the fractional changes in fluorescence relative to the background fluorescence level. This does not, however, eliminate the signal variability completely, because the fractional change of fluorescence itself may vary throughout the preparation (mainly due to nonuniform dye internalization).

In defibrillation studies, the Vm-independent variability can be eliminated by normalization of optical signals relative to their respective action potential amplitudes (APA).23 This procedure is especially useful in measuring shock-induced Vm changes (AVm) that are typically normalized by the APA values. Such signal normalization is based on the assumption that APA does not change across the imaged area, which is likely to be true in healthy, well-coupled tissue. The validity of this condition is even more likelier in microscopic measurements, when the size of an imaged area is comparable to the length of the electrotonic constant, which in cell monolayers is about 360 ^m.24 This assumption might not hold true on a larger spatial scale in pathologic conditions, such as ischemia, which leads to nonuniform distribution of APA.

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