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Because gene therapy presents a new frontier in the treatment of arrhythmias, it has become important to know how manipulation of the cellular distribution of proteins changes electrical events within individual cells, and whether these cellular changes affect conduction at the larger macroscopic size scale. However, experimental limitations in cardiac bundles prevent measurement of conduction delays across specific gap junctions, as well as the intracellular distribution of the maximum rate of rise of the action potential (V(max)). In view of these limitations, we used immunohistochemical morphological results as a basis to develop two-dimensional cellular models of neonatal and mature canine ventricular muscle in order to obtain insight into the electrophysiological effects of changes in the cellular distribution of proteins; eg, the major protein of cardiac gap junctions, connexin43. Morphological results showed that when the cells enlarged after birth, the gap junctions shifted from the sides to the ends of ventricular myocytes. At birth, V(max) was not different during longitudinal and transverse propagation. However, growth hypertrophy produced a selective increase in mean transverse V(max) with no significant change in longitudinal V(max). Two-dimensional cellular computational models of neonatal and mature ventricular muscle showed that the observed changes in the cellular distribution of the gap junctions and change in cell size accounted for the experimental results. The results unexpectedly showed that cellular scaling (cell size) is as important (or more so) as changes in gap junction distribution in determining the properties of transverse propagation. The results suggest that in pathological states that are arrhythmogenic, maintenance of cell size during remodeling the distribution of gap junctions is important in sustaining a maximum rate of rise of the action potential.

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The increased incidence of arrhythmias in structural heart disease is accompanied by remodeling of the cellular distribution of gap junctions to a diffuse pattern like that of neonatal cardiomyocytes. Accordingly, it has become important to know how remodeling of gap junctions due to normal growth hypertrophy alters anisotropic propagation at a cellular level (V(max)) in relation to conduction velocities measured at a macroscopic level. To this end, morphological studies of gap junctions (connexin43) and in vitro electrical measurements were performed in neonatal and adult canine ventricular muscle. When cells enlarged, gap junctions shifted from the sides to the ends of ventricular myocytes. Electrically, normal growth produced different patterns of change at a macroscopic and microscopic level. Although the longitudinal and transverse conduction velocities were greater in adult than neonatal muscle, the anisotropic velocity ratios were the same. In the neonate, mean V(max) was not different during longitudinal (LP) and transverse (TP) propagation. However, growth hypertrophy produced a selective increase in mean TP V(max) (P<0.001), with no significant change in mean LP V(max). Two-dimensional neonatal and adult cellular computational models show that the observed increases in cell size and changes in the distribution of gap junctions are sufficient to account for the experimental results. Unexpectedly, the results show that cellular scaling (cell size) is as important (or more so) as changes in gap junction distribution in determining TP properties. As the cells enlarged, both mean TP V(max) and lateral cell-to-cell delay increased. V(max) increased because increases in cell-to-cell delay reduced the electric current flowing downstream up to the time of V(max), thus enhancing V(max). The results suggest that in pathological substrates that are arrhythmogenic, maintaining cell size during remodeling of gap junctions is important in sustaining a maximum rate of depolarization.

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It has become of fundamental importance to understand variations in the shape of the upstroke of the action potential in order to identify structural loading effects. One component of this goal is a detailed experimental analysis of the time course of the foot of the cardiac action potential (Vm foot) during propagation in different directions in anisotropic cardiac muscle. To this end, we performed phase-plane analysis of transmembrane action potentials during anisotropic propagation in adult working myocardium. The results showed that during longitudinal propagation there was initial slowing of Vm foot that resulted in deviations from a simple exponential; corollary changes occurred at numerous sites during transverse propagation. We hypothesized that the effect on Vm foot observed in the experimental data was created by the microscopic structure, especially the capillaries. This hypothesis predicts that the phase-plane trajectory of Vm foot will deviate from linearity in the presence of a high density of capillaries, and that a linear trajectory will occur in the absence of capillaries. Comparison of the results of Fast and Kléber (Circ Res. 1993;73:914-925) in a monolayer of neonatal cardiac myocytes, which is devoid of capillaries, and our results in newborn ventricular muscle, which is rich in capillaries, showed drastic differences in Vm foot as predicted. Because this comparison provided experimental support for the capillary hypothesis, we explored the underlying biophysical mechanisms due to interstitial electrical field effects, using a "2-domain" model of myocytes and capillaries separated by interstitial space. The model results show that a propagating interstitial electrical field induces an inward capacitive current in the inactive capillaries that causes a feedback effect on the active membrane (source) that slows the initial rise of its action potential. The results show unexpected mechanisms related to extracellular structural loading that may play a role in selected conduction disturbances, such as in a reperfused ischemic region surrounded by normal myocardium.

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The purpose of this article is to demonstrate how adaptive changes in myocardial microstructure provide mechanisms for emergent new conduction disturbances that initiate reentrant arrhythmias. The mechanisms are based on discontinuous conduction phenomena produced by increases in cellular loading; these increases result from changes in the normal distribution of the gap junctions. Recent studies that at a microscopic level propagation in normal mature cardiac muscle is stochastic. For example, the nonuniform and irregular distribution of the gap junctions in such normal muscle produces load variations that are associated with changes in Vmax inside individual cells during both longitudinal and transverse propagation. The stochastic nature of normal propagation at a microscopic level offers considerable protection against arrhythmias by reestablishing the general trend of wavefront movement after small variations in excitation events occur. If such microscopic diversity is decreased, large fluctuations in load develop that are distributed over more cells than usual. The decrease in diversity may be caused by loss of side-to-side coupling between fibers, which produces relatively isolated groups of cells with microfibrosis. With loss of side-to-side fiber coupling, the myocardial architecture may fail to reestablish a smoothed wavefront at the macroscopic level. Spatial nonuniformities of electrical loading then give rise to conduction block and reentry.

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INTRODUCTION: AV nodal reentrant tachycardia cycle length has been shown to be longer in the elderly population. Microfibrosis associated with aging producing nonuniform anisotropic conduction or changes in membrane ionic properties could explain this finding. METHODS AND RESULTS: Forty-five patients (33 women and 12 men) with typical AV nodal reentrant tachycardia were studied to analyze the effects of age on electrophysiologic characteristics of the tachycardia using high-density catheter mapping of the triangle of Koch. We classified patients into group A (age < or = 45 years, mean [+/-SD] 32.7 +/- 8.8, n = 27) and group B (age > 45 years, mean [+/-SD] 61.1 +/- 10.2, n = 18). Retrograde atrial activation was recorded during tachycardia by means of a 2-mm decapolar catheter at the His bundle, a quadripolar catheter at the high right atrium, a multipolar catheter (6 to 10 poles) in the coronary sinus, and a deflectable quadripolar catheter at the posterior triangle of Koch. The AH interval at the AV junction as well as HA intervals at several atrial sites were measured during tachycardia. HA intervals at all atrial recording sites except in the posterior triangle of Koch were significantly longer in group B, as well as the tachycardia cycle length (362 vs 329 msec, P = 0.01). The mean AH interval was prolonged by 24 msec in group B, but this difference did not reach statistical significance. A sequential pattern of retrograde atrial activation during tachycardia was more frequently recorded in group B. CONCLUSIONS: Since the delayed activation to the atrium was heterogeneous, transverse nonuniform anisotropic conduction is a likely explanation of these age-related modifications of AV nodal reentrant tachycardia characteristics.

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The object of this study is to present evidence that the myocardial architecture creates inhomogeneities of electrical load at the cellular level that cause cardiac propagation to be stochastic in nature; ie, the excitatory events during propagation are constantly changing and disorderly in the sense of varying intracellular events and delays between cells. At a macroscopic level, however, these stochastic events become averaged and appear consistent with a continuous medium. We examined this concept in a two-dimensional (2D) model of myocardial architecture by exploring whether experimentally observed Vmax variability reflected different patterns of intracellular excitation events and junctional delays. The patterns of Vmax variability at randomly chosen intracellular sites were similar experimentally and in the 2D model. The 2D cellular model produced marked variability in gap junction delays; however, on the average, different gap junctions were used for cell-to-cell charge flow during conduction in different directions. During longitudinal propagation (LP), the velocity increased from the proximal to the distal end of each myocyte, and Vmax was lowest proximally, increased to a maximum at the distal fourth of the cell, and decreased distally. Transverse propagation (TP) produced rapid intracellular conduction with variable intracellular excitation sequences. TP Vmax was greater than LP Vmax in most subcellular regions, but near the ends of some myocytes, a reversed "TP > LP Vmax" relation occurred. Total charge carried by the sodium current varied inversely with Vmax, demonstrating feedback effects of cellular loading on the subcellular sodium current and the kinetics of the sodium channels. The results suggest that the stochastic nature of normal propagation at a microscopic level provides a considerable protective effect against arrhythmias by reestablishing the general trend of wave-front movement after small variations in excitation events occur.

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To study the spread of excitation in ventricular heart preparations we have designed a fast, high-resolution recording and mapping system. Papillary muscles were dissected from the isolated guinea pig hearts. The preparation was fixed in a tissue bath and superfused with Tyrode solution. Linear and two-dimensional arrays of Ag/AgCl electrodes were made on glass with a thin-film technique. The transparent sensors with up to 24 electrodes (spaced 50, 90, or 180 microns apart) were positioned close to the surface of the preparation with a custom-designed three-dimensional micromanipulator. Extracellular signals were simultaneously recorded by a 24-channel data acquisition system with a 200 kHz per channel sample rate, with 12-bit amplitude resolution and a maximum data length of up to 3 MB. Digitized video images of the electrode array and the underlaying preparation were used to identify the locations of the recording sites. A UNIX-based computer system with a custom-designed data acquisition and database program was used to control the instruments and to manage the experimental data. This technique gave signals with excellent signal-to-noise ratios (up to 65 dB) and permitted accurate evaluation of the time and the site of the local activation with high resolution (to within 5 microseconds, 50 microns). We describe the spread of excitation within the area of a few cells and found a substantial dispersion of conduction velocities. Beat-to-beat comparison of activation patterns showed relatively small variations in the spread of excitation (a few microseconds).

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Until recently only two types of media have been considered to provide the nonuniformities necessary to initiate cardiac reentry: (1) continuous isotropic media with intrinsic repolarization inhomogeneities; and (2) continuous isotropic media free of inhomogeneities in which repolarization nonuniformities are introduced transiently. The purpose of this article is to establish cellular coupling as a basis for arrhythmias by placing a new type of inhomogeneity, nonuniform anisotropy due to sparse side-to-side coupling between cells, in an overall perspective with the other nonuniformities that lead to reentry. Review of experimental and theoretical models of reentry leads to the following picture: with slowed conduction, reentrant circuits diminish in size and the nonuniformities necessary for reentry are provided by nonuniform anisotropy. Repolarization nonuniformities create functionally different pathways for reentrant circuits of relatively large size (> 30-50 mm2). Nonuniform anisotropic cellular coupling, which is associated with underlying microfibrosis, makes it possible for reentry to occur in small areas (< 10-15 mm2). A general property found in nonuniform anisotropic bundles is the presence of functionally different pathways in the absence of intrinsic repolarization inhomogeneities--one of fast longitudinal conduction with a longer refractory period, and another of "very slow" transverse conduction with a shorter refractory period. Since it is not known if nonuniform anisotropy exists in the AV node, the best known structure with small reentrant circuits, we performed microscopic extracellular measurements in the AV node of the rabbit. The transitional zone of the AV node was found to have markedly nonuniform anisotropic conduction properties. The analysis provides the view that functionally different pathways of small reentrant circuits, including those of the AV node, need to be reevaluated in terms of the role of nonuniform anisotropic cellular coupling.

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The arrangement of collagen fibers has previously been studied with picrosirius red (PSR) staining and brightfield microscopy. We discovered that PSR staining can also be visualized by fluorescence microscopy. PSR-stained collagen was strongly fluorescent using excitation and barrier filters for rhodamine, and distracting background cytoplasmic fluorescence was drastically reduced with phosphomolybdic acid (PMA) treatment before PSR staining. The PMA-PSR fluorescence method was more sensitive than the brightfield PSR or PMA-PSR method, and permitted confocal microscopic study. We applied the method to the study of collagen fiber three-dimensional arrangement in perimysial and endomysial septa of the heart, showing the three-dimensional course of the fibers in stereo views generated by confocal microscopy. The PMA-PSR fluorescence method should be generally useful for accurately determining collagen fiber three-dimensional arrangement, a necessary prelude to mechanical modeling of collagen-reinforced tissues.

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This study was designed to test the hypothesis that the electrical load seen at a microelectrode impalement site is sensitive to the direction of propagation of the approaching wavefront as a reflection of an altered spatial relationship between the impalement site and the surrounding microscopic electrical boundaries located up- and downstream. These boundaries correspond to the different sizes and shapes of the impaled and surrounding cells as well as to the distribution of the associated electrical connections between the cells. The effects of changes in these geometric relationships on maximum rate of rise of transmembrane potential (Vmax) were investigated in canine ventricular muscle by measuring Vmax in different cells while the direction of propagation was changed from along the longitudinal axis to the transverse axis of the fibers or the direction of conduction was reversed along either of these axes. Comparison of the Vmax values for longitudinal propagation (LP) and transverse propagation (TP), each in one direction, showed that TP Vmax was significantly greater than LP Vmax (P < 0.001). However, the values of Vmax were different from cell to cell during LP (93-139 V/s) and TP (110-181 V/s). The absolute values of LP Vmax and TP Vmax at the same site varied independently of each other, e.g., some of the lowest LP Vmax values occurred at the same site as the highest TP Vmax values. Furthermore, at the same site, Vmax changed considerably when propagation was maintained along the longitudinal axis but the direction of conduction was reversed. Similar prominent changes in Vmax occurred when the direction was reversed along the transverse axis.(ABSTRACT TRUNCATED AT 250 WORDS)

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To assess the distribution of gap junctions in relation to the cardiac myocyte surface in paraffin sections of dog and rat ventricle, the sarcolemma was labeled with wheat germ agglutinin (WGA1) and gap junctions were labeled with antibodies to cardiac muscle gap junction protein connexin43. WGA labeled all of the myocyte sarcolemma, including that in intercalated discs and transverse tubules. Sarcolemmal WGA labeling was often interrupted at the sites of gap junctions, which were found both at the extreme ends of myocytes and along the length of adjacent myocytes. Small gap junctions predominated at plicate transverse portions of the intercalated disc; larger and sometimes ribbon-like gap junctions predominated at longitudinal portions. The longitudinal portions of the intercalated disc often extended over multiple sarcomere lengths, with ribbon-like gap junctions and linear arrays of smaller gap junctions arranged in parallel overlying successive sarcomeres. Morphometric study showed that ribbon-like gap junctions were relatively infrequent in both dog and rat left ventricular epimyocardium, and that animals with larger myocytes tended to have smaller gap junctions. In dog left ventricular epimyocardium, neither myocytes nor their larger gap junctions were randomly oriented with respect to perimysial separations; myocytes were usually somewhat flattened with their maximal diameters parallel to the separations, whereas large gap junctions were least often oriented parallel or perpendicular to the separations. Overall, the data indicate that myocyte geometry influences gap junction size and distribution; the double-label technique is ideally suited for the further exploration of that influence.

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This study was designed to develop a two-dimensional cellular model of uniform anisotropic muscle and to determine how irregularities of shape and variations in size of cardiomyocytes influence the passive (electrotonic) spread of currents at a microscopic level. A secondary purpose was to determine how the passive transfer of impressed currents across the gap junctions is related to the charge flow across the gap junctions during active propagation of depolarization. The decrease in electrotonic Vm with distance at a large size scale was described by a single exponential in both the longitudinal and transverse directions, as occurs in a continuous anisotropic medium. At a microscopic level, however, the falloff of Vm with distance was directionally different. Longitudinally, Vm decreased primarily along the length of cells, with small step-like decreases at the intercalated disks. Transversely, Vm was more nearly isopotential throughout each cell, and most of the decay in Vm occurred as large step-like decreases across the borders of the cells. Different gap junctions were used for charge flow for longitudinal versus transverse electrotonus. Remarkably similar results were obtained for propagating action potentials, i.e., different gap junctions were used for longitudinal versus transverse conduction. A major implication of the results is that it may be possible to gain information about the different longitudinal and transverse effects of the nonhomogeneous distribution of the cellular connections by improved measurements of propagation at a microscopic level.

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Recent studies of isolated cardiac myocytes have generated the need for detailed information about regional electrophysiological differences in the atrium. We measured the spatial distribution of action potentials in adult and newborn canine right atria. Multiple regional differences in action potential shape and duration were found. The multiple regional differences produced an overall simple pattern: the longest action potentials occurred in the area of the sinus node, and the action potential duration decreased with increasing distance from the sinus node area. To account for the overall pattern, we tested factors considered important in causing atrial action potential shape differences (e.g., electronic interactions). None of the factors tested accounted for the regional differences. We then found regional differences in the responses to pauses, which suggested that differences in the properties of individual cells accounted for the regional repolarization differences. If so, genetic regulation of the regional differences may produce the overall pattern on a developmental basis. Experiments in newborn atria showed that only in the upper crista was the spatial pattern similar to that of the adult; there was little variability in action potential shape and duration in the other areas. As a further test for associated regional differences in cell properties, we examined for differences in the isoform expression of troponin T (TnT1, TnT2, TnT3, and TnT4), a protein important in excitation-contraction coupling. In adults, the greatest proportion of TnT1 occurred in the area of the sinus node, and its proportion decreased with increasing distance from the sinus node area in association with a relative increase in the proportion of TnT2. In newborn atria the relative amount of TnT1 was greatest in the upper crista (similar to adult), but little difference was found in the distribution of the isoforms in the other regions. The correspondence between the regional differences in repolarization and in the expression of the troponin T isoforms in adult and newborn atria suggests that 1) cellular ionic mechanisms vary regionally to coordinate differences in action potential configuration with differences in cell properties that regulate contractility and 2) genetic expression of the systems that regulate repolarization and mechanical cellular properties are under similar developmental and regional control.

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