Cardiac Gap Junctions: Physiology, Regulation, Pathophysiology and Pharmacology

Cardiac Gap Junctions Physiology, Regulation, Pathophysiology and Pharmacology S. Dhei...

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Cardiac Gap Junctions Physiology, Regulation, Pathophysiology and Pharmacology

S. Dhein, Cologne

23 figures and 3 tables, 1998

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Stefan Dhein Institute of Pharmacology University of Cologne (Germany)

All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Ó Copyright 1998 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) Printed in Switzerland on acid-free paper by Reinhardt Druck, Basel ISBN 3–8055–6567–4

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Dedicated to Aida

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Contents

Foreword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XI 1 2 3 4 5 6 7 8

Introduction: Cellular Coupling, Cardiac Activation Patterns and Arrhythmia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure and Diversity of Gap Junction Channels . . . . . . . . . Distribution of Gap Junctions in the Heart . . . . . . . . . . . . . Function and Physiology of Gap Junction Channels . . . . . . . . Regulation of Gap Junction Expression, Synthesis and Assembly Gap Junctions in Cardiac Disease . . . . . . . . . . . . . . . . . . . Pharmacological Interventions at Gap Junctions . . . . . . . . . . Methods for Investigation of Gap Junctions . . . . . . . . . . . . .

. 1 . 13 . 25 . 35 . 63 . 73 . 89 . 106

References . . . . . . . . . . . . . . . . . Appendix . . . . . . . . . . . . . . . . . List of Suppliers of Specialized Items Subject Index . . . . . . . . . . . . . . .

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VII

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Foreword

When I started as a novice in the field of cardiac electrophysiology, the dogma was that gap junctions are specialized membrane structures present in the cardiac and smooth muscle of vertebrates where they serve to propagate the action potential from cell to cell. Purkinje fibers and muscular trabeculae were the preferred cardiac preparations. These multicellular preparations were suitable to perform cable analyses and diffusion studies. At that time, my mentor, Silvio Weidmann, had already accomplished his elegant functional studies. The subsequent progress in the field was prompted largely by the development of novel experimental approaches. On the one hand, the introduction of the patch-clamp method and the use of cell pairs led to a detailed description of the intercellular current flow. As a result, we nowadays have extensive knowledge about the conductive and kinetic properties of gap junctions and gap junction channels. On the other hand, immunohistochemistry and molecular biology made one aware of the diversity of gap junction proteins and their distribution in tissues of the cardiovascular system. This reductionistic approach led to the accumulation of an enormous amount of functional and structural details. The combination of electrophysiology and molecular biology will culminate eventually in the elucidation of the structure-function relationship of a single channel. However, scientists soon should explore the reverse path and try to integrate the collected data in the context of an intact heart. In this way, the knowledge gathered may provide a basis for new strategies against cardiovascular diseases. Hopefully, this monograph will contribute to this process. Robert Weingart Berne, June 23, 1997

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Preface

‘Cells live together and die singly’ Engelmann wrote at the end of the past century. With this simple sentence he summarized the key feature provided by cell coupling via gap junction channels: these channels provide exchange of small molecules and electrical coupling in the intact heart, but in the course of ischemia, for example, they close, the cell gets isolated and is no longer activated by the surrounding tissue. This may help the cell to survive, or the cell dies but without influencing the adjacent cells. In the chronic phase of cardiac disease the distribution of various gap junction isoforms can change, thereby altering the tissue’s biophysics. This behavior opens new perspectives for arrhythmia research, for drug research and for research directed toward ischemia, cardiac protection and cardiac pathophysiology. The following book is written to give an insight into a relatively new field of cardiovascular research to basic researchers, cardiologists, physiologists and pharmacologists who wish to obtain information on cellular coupling in the heart or who wish to enter this new field of research. Therefore, the first chapter gives an introduction to the various aspects of activation propagation and coupling in the heart. This is followed by two chapters which review our present knowledge on the structural aspects of gap junction channels including amino acid sequences and species variability as known so far. Thereafter, the physiology of gap junction channels and the regulation of expression is described in the subsequent two chapters. Since it is known today that gap junction distribution can change in the course of cardiac disease, these changes and their implications are described in the sixth chapter. The seventh chapter then gives insight into pharmacological approaches to the modulation of gap junction channel conductivity and outlines possible new therapeutic strategies. The final chapter is especially written for people who are interested in entering this fascinating field of cardiovascular research and describes practical approaches to gap junction research. The concept of double-cell voltage clamp, immunohistochemistry, isolation procedures for gap junctions and dye-coupling assays are described with practical protocols. At the end of the book a list of suppliers of specialized items, such as certain amplifiers, antibodies etc., is given. Stefan Dhein Cologne, April 1997

XI

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Introduction: Cellular Coupling, Cardiac Activation Patterns and Arrhythmia

What is the reason for thinking about cardiac gap junctions? In the last years, after the cardiac arrhythmia suppression trial (CAST), the understanding of cardiac arrhythmia and antiarrhythmic drug therapy has completely changed. In that study Echt et al. [1991] observed lethal arrhythmia in patients under antiarrhythmic treatment with class-Ic agents (flecainide) during the postinfarction period. Until that study, proarrhythmic drug activity had been widely neglected although it was reported earlier [Brugada and Wellens, 1988; Podrid, 1985; Podrid et al., 1987]. According to these studies and CAST, it can be concluded that prophylactic treatment with antiarrhythmic drugs can paradoxically induce arrhythmia or aggravate arrhythmia in therapeutic concentrations. This has been defined as the proarrhythmic risk of antiarrhythmic drugs. First, it was confined to class-I antiarrhythmic agents but in between it became evident that not only class-I antiarrhythmics but also class-III antiarrhythmics exhibit proarrhythmia, the latter especially as torsade de pointes arrhythmia [Carlsson et al., 1990]. In in vitro studies using isolated rabbit hearts, it was shown that all class-I antiarrhythmics induced significant alterations in the activation patterns and disturbed the normal excitation process under control conditions [Dhein et al., 1993b]. Thus, it was concluded by these investigators that prophylactic treatment with antiarrhythmic drugs may disturb the normal excitation process thereby inducing alterations in the geometry of the activation pattern, which finally lead to arrhythmia. What were the consequences? Prophylactic antiarrhythmic drug treatment has changed to a therapy which is carried out with great caution and care. The surprising finding that antiarrhythmic drugs can provoke arrhythmia made it evident that arrhythmia is not only a phenomenon of a single cell but involves the whole tissue and that more determinants are involved than only the transmembrane currents. The term, arrhythmogenic substrate, became a matter of interest to many researchers. The arrhythmogenic substrate means the pathologic and anatomic preconditions for the initiation of tachyarrhythmias such as myocardial fibrosis, aneurysm, the border zone between normal and ischemic or infarcted tissue, scars, diffuse myocardial injury in cardiomyopathy or the chronic alterations induced by myocarditis, and furthermore, accessory pathways or variations in the specific cardiac conduction system. These anatomic or pathologic altera-

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tions alone do not provoke arrhythmia, but if additional factors, such as variations in the autonomous innervation, changes in the electrolyte balance, extrasystoles or changes in the pacing frequency, coincide, these factors work in concert with the pathological and anatomical alterations and finally cause arrhythmia. In consequence, the goal of many studies was to define the changes responsible for arrhythmogenesis on the tissue or whole organ level. Many investigators sought for new approaches to arrhythmogenesis and antiarrhythmic agents. Especially safe antiarrhythmic agents for use in prophylactic treatment are searched for. In the course of this development focusing on the arrhythmogenic substrate on the basis of tissue alterations the question how myocardial cells interact with each other became the center of attention. The intercellular communication via intercellular low-resistance pathways (gap junctions), other forms of coupling and the cardiac networking became an important subject of research. How are cardiac cells coupled? How do cardiac cells interact electrically? These are important questions to be addressed and they lead to the topic of cardiac networking. Sperelakis [1979] distinguished three forms of transfer of excitation: (1) mechanical transmission; (2) chemical transmission, and (3) electrical transmission. Mechanical transmission, i.e. the contraction of the pre-cell depolarizes the membrane of the post-cell via membranous stretching, can be ruled out as an important mechanism in the heart because the electromechanical coupling time in the heart is longer than the time available for the transfer. Chemical transmission has been postulated with K+ as transmitter, since the K+ effluxing during an action potential will diffuse rapidly in the bulk interstitial fluid around the sarcolemma but can accumulate in the narrow cleft of the intercalated disk thereby depolarizing the post-membrane. This might contribute to the transmission process [Macdonald et al., 1975]. However, the most important transfer mechanism is electrical transmission via low-resistance pathways, which have been identified as gap junction channels. An early argument in support of the concept of low-resistance pathways was that the length constant k (measured in cardiac muscle bundles by extracellular application of current) ranges between 0.5 and 2.0 mm and that the input resistance (measured change in voltage at the site of current injection divided by the applied current) is comparably low indicating that current passes to neighboring cells. Besides this, capacitive coupling and electrical field coupling have been proposed as alternative mechanisms of electrical transmission. Capacitive coupling according to Sperelakis [1979] means that a capacitive current flows through a capacitance (the membrane is a capacitor and the action potential is an alternating current AC signal) that acts to couple

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Introduction

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both cells. For several physical reasons (junctional capacitive coupling would be decreased by a factor of 2 since the junction resembles 2 capacitors in series; next, if only a small portion of the intercalated disk is involved, the total capacitance would be accordingly smaller, and third there may be a shunt to ground if the two membranes are not close enough to each other), capacitive coupling may not work in normal cardiac tissue [for a detailed discussion of that matter see, Sperelakis, 1979]. Electrical field coupling [Sperelakis and Mann, 1977] means the induction of an action potential in the post-cell by the electrical field arising from the action potential at the intercalated disk of the pre-cell. The authors showed that an accumulation of K+ in the cleft of the intercalated disk is an important contributory factor allowing the membrane of the pre-cell at the intercalated disk to fire a fraction of a millisecond earlier than the surface membrane, which was necessary for effective coupling. However, at present it is uncertain what the contribution of electrical field coupling to electrical transmission is in normal tissue. Weingart and Maurer [1988] showed that after manipulating two separate cardiac ventricular cells into intimate side-to-side contact initially, i.e. before forming gap junction channels, there was no transmission of electrotonic potentials or action potentials from one cell to the other. This experiment is against the theory of ephaptic impulse transmission or of electrical field coupling [Sperelakis, 1979] as a non-gap junctional mechanism of intercellular action potential spreading. However, it remains to be elucidated whether more tissue than only one cell is needed for electrical field coupling or capacitive coupling. The mass of activated tissue might be a determining feature. The composition of the interstitial fluid, the K+ concentration in the clefts, and the geometry of the clefts between two adjacent cells may also contribute to the local electrical properties. In addition, it can be imagined that in tissue, if gap junctions close and the low-resistance pathways are occluded, these forms of coupling may become more important. In summary, from the present point of view the most important mechanism for transmission of excitation is coupling via the gap junction channels. Considering the passive electrical properties of the tissue means first of all to consider the properties of a muscle bundle, i.e. the passive cable properties. Muscle fibers are classically considered cables consisting of cells coupled in series via ohmic resistors with each cell representing a resistor with a parallel capacitor [for review see, Weidmann, 1990]. The change in voltage is a function of distance (x) according to Vx> Vx0(expÖx/k) with the length constant k>z(rm /ri ) (rm>membrane resistance, ri>internal longitudinal resistance); the input resistance at x>0 can be described as rinput>Vx0 /I>ri k. Taking the fiber radius into account, the specific membrane resistance Rm equals 2parm [Xcm2] and specific internal resistance Ri>pr2ri . With the specific membrane

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Introduction

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capacitance the time constant s is described as s>CmRm. In a multicellular preparation with parallel running fibers the longitudinal resistance of the extracellular space ro also has to be considered. For these conditions k is reflected by k>z(rm /[ri+ro]) and the conduction velocity h depends on h>z(1/{TfootCm[ri+ro]}). However, this cable theory, originally formulated for nerve axons [Hodgkin and Rushton, 1946] and later on for Purkinje fibers [Weidmann, 1952], is based on the assumption of continuity of the cable. In consequence of these assumptions, passive membrane properties were considered to be of minor importance for the pathophysiology of conduction disturbances and the basic mechanisms were ascribed to membrane ionic properties and their regional differences. However, in cardiac tissue the situation is a bit more complicated by anisotropy and non-uniformity. Basically, action potential propagation is faster along the longitudinal axis of the fibers than in the transverse direction due to higher intercellular resistance perpendicular to the fiber axis. In addition, on a microscopic basis propagation is discontinuous and the inhomogeneous and anisotropic distribution of the cellular connections influence action potential upstroke and the safety factor of propagation [Spach and Dolber, 1990]. The difference between the two forms of anisotropy, i.e. uniform versus nonuniform, has many consequences for the pathophysiology of arrhythmia. First of all, it was observed that the action potential upstroke velocity and amplitude were greater during transverse propagation. This was accompanied by a faster foot potential and led to the hypothesis that longitudinal propagation is, although faster, more vulnerable to block because of its lower upstroke velocity and amplitude. This behavior can be explained on a theoretical basis: the upstroke velocity increases as a result of reduced coupling [Delmar et al., 1987] since the current can not pass to the neighboring cells. In nonuniform anisotropic tissue fractionated extracellular waveforms are often encountered. Such complex waveforms with multiphasic shape can be interpreted as the reflection of discontinuous propagation and each of the multiple negative peaks represent the activation of a small group of fibers. It should be kept in mind that with aging there is a general change in the biophysical properties of the cardiac tissue from uniform to nonuniform anisotropy due to predominant uncoupling of side-to-side connections with increasing age [Spach and Dolber, 1986, 1990]. What are the consequences of nonuniformity for action potential propagation? In fibers with tight electrical coupling, simulated extrasystoles with the shortest interval leading to a propagated response lead to a progressive reduction in conduction velocity in all directions (i.e. in parallel to the reduced sodium channel availability). In contrast, in nonuniform tissue the earliest premature beat leads to dissociated microscopic longitudinal propagation, i.e. the large biphasic waveform changes

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Introduction

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to a multiphasic fractionated one. Similar behavior in nonuniform anisotropic tissue could be induced with use-dependent sodium channel-blocking agents (e.g. quinidine). Besides this, more importantly, early premature beats in nonuniform anisotropic tissue can induce conduction block in longitudinal direction while transverse propagation is still possible indicating a lower safety factor for longitudinal propagation in this tissue. It has been shown that this situation can initiate reentrant arrhythmia [Spach and Dolber, 1990; Spach et al., 1988]. What is the basis? According to the leading circle concept [Allessie et al., 1977] it could be argued that the premature beat encounters refractory tissue. However, the authors could demonstrate even lower refractory periods at the site of conduction block, and conclude from their findings that the microreentry was solely based on discontinuous anisotropic propagation. The safety factor for longitudinal propagation is reduced when sodium conductance is decreased as the consequence of early premature beats. Under these conditions the discontinuities in nonuniform anisotropic tissue can cause longitudinal conduction block, whereas in homogeneous, i.e. uniform, anisotropic tissue block occurs in both directions [for a detailed discussion see, Spach and Dolber, 1990]. It is important to stress the point that on a macroscopic scale (many millimeters) propagation may behave as in continuous tissue, but on a microscopic scale discontinuous propagation occurs and this discontinuous propagation can cause slow conduction of even normal action potentials. Thus, slow conduction does not necessarily mean depressed conduction [Spach and Dolber, 1990]. What are the effects of coupling itself on transverse and longitudinal propagation? Delmar et al. [1987] investigated longitudinal and transverse propagation in thin layers of sheep cardiac muscle before and after superfusion with heptanol, an agent which reduces gap junctional coupling (see chapter 7). They found out that transverse propagation is more sensitive to electrical uncoupling indicating a lower safety factor under these circumstances. After exposure to heptanol, conduction block in transverse direction occurred after about 28 min whereas longitudinal block was observed after about 44 min. With regard to the findings of Spach and coworkers considered above, the authors suggested that uncoupling can have opposite directional effects to those seen if sodium conductance is reduced. It might be speculated that the smaller number of gap junctions at the side-to-side border as compared to the intercalated disks might form the basis for a higher sensitivity of transverse propagation to uncoupling. In summary, longitudinal propagation seems to be more sensitive to reduced sodium channel availability especially in nonuniform anisotropic tissue, and under these conditions reentrant arrhythmia can be initiated due to discontinuous propagation, whereas transverse propagation is more sensitive to un-

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Introduction

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coupling. However, it should be taken into account that heptanol does not specifically block gap junctions and that under most pathophysiological conditions, for example ischemia, complex changes occur with at least both reduced sodium channel availability and gap junctional uncoupling. In such complex situations it is probably difficult to foresee whether longitudinal or transverse propagation will fail. Anisotropy and nonuniformity are at least in part due to inhomogeneities in the distribution of gap junctions and the biophysical properties of the tissue are in fact influenced by the intercellular coupling. At least four features have to be considered. (1) Cardiac cells express different gap junction proteins (socalled connexins; in the heart, connexin 40, connexin 43 and connexin 45 are most abundantly found; for details see chapters 2 and 3). Channels formed by these connexins are different with regard to their biophysical properties. In various parts of the heart the content of each of these isoforms is different. (2) There is a highly distinctive three-dimensional spatial distribution of intercellular connections with different patterns in different parts of the heart. (3) Furthermore, it has been shown that a single cardiomyocyte can express different isoforms of connexins [Saffitz et al., 1995]. Thus, the formation of heterotypic channels combining the biophysical properties of more than one connexin is possible. (4) In the course of cardiac disease the specific pattern of gap junction distribution can be altered, thus inducing changes in the conduction properties of the tissue (see chapter 6). Taken together, there are a large number of putative mechanisms regulating and modulating intercellular communication in the heart. The anisotropic ratio varies between different parts of the heart. In the crista terminalis for example the ratio between the longitudinal and transverse propagation velocity is 10:1, whereas in ventricles the ratio was found to be 3:1. What is the basis of this phenomenon? Saffitz et al. [1994] showed that in the crista terminalis each cell is connected to 6.4×1.7 other cells whereas in the ventricle each cell is connected to 11.3×2.2 other myocytes. In addition, in the crista terminalis the gap junctions were confined to the ends of the cells forming predominantly end-to-end-connections whereas in the ventricles approximately similar numbers of gap junctions occurred in end-to-end and side-to-side orientation. Thereby, the effective length to width ratio of a ventricular cell is reduced from 6:1 to 3.4:1 [Saffitz et al., 1995], thus lowering the degree of anisotropy. Due to this complex architecture, in the ventricle a wavefront encounters a broad spectrum of possibilities to propagate in a longitudinal or transverse direction. Moving in the transverse direction, however, makes it necessary to pass over more intercellular connections, which means a higher resistance in that direction, so that the wavefront is slowed. In some cardiac diseases, e.g. in the border zone of healed infarction, the gap

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Introduction

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junction distribution is changed, each cell is connected to a fewer number of other cells and especially the side-to-side connections are reduced [Luke and Saffitz, 1991] (see chapter 6). As a consequence, a wavefront travelling transverse to the fiber axis becomes slowed specifically in this critical area, whereas it propagates with normal velocity in the surrounding normal tissue. In this border zone this transversely propagating wavefront has to follow the sparse side-to-side connections and, thus, is urged to zigzag through the zone. This enhances the possibility of the wavefront meeting postrefractory tissue and initiating the next beat of tachycardia. This demonstrates how the architecture and structure of the tissue, the passive properties of the tissue and the intercellular coupling pattern can contribute to both the physiological activation pattern and the pathophysiological alterations in the activation pattern, and thus are an important determining factor in arrhythmogenesis. These factors have long been neglected, cardiovascular research was mainly focused on the active membrane properties of single cells. At the microscopic level, cardiac myocytes are shaped irregularly and gap junctions are distributed in a nonuniform manner (see chapter 3). Because the direction of propagation varies, Spach and Heidlage [1995] investigated the implications of these microscopic irregularities for the load variations within individual cells. They modelled a two-dimensional array of cells (each cell consisting of a finite number of 10 * 10-lm segments) with plicate junctions (in the plicate segment of the intercalated disk), interplicate junctions (in regions close beside the plicate segments) and combined plicate junctions (small step-like irregularities at the cell border) assuming the gap junctions to behave as ohmic resistors of 0.5 (plicate), 0.33 (interplicate) and 0.062 lS (combined plicate junction). They found that during longitudinal propagation the upstroke velocity of the local action potential Vmax was lowest at the proximal end of the cell, increased to its maximum at the distal fourth and decreased distally. During transverse propagation, higher Vmax as well as rapid intracellular conduction with variable intracellular pathways was observed. At the end of some myocytes higher Vmax was found for transverse propagation. The charge elicited by the fast sodium current was inversely related to Vmax. The surrounding cellular network exhibited a strong modulating influence on gap junction delay, Vmax and sodium current. Coupling the cells to their surrounding resulted in a decrease in Vmax, an increase in gap junction delay and in sodium current. Discontinuities during longitudinal propagation were observed at the end-to-end connections of the cells and, during transverse propagation, ‘large lateral jumps’ were found which coincided with the lateral borders of the cells. In summary, they concluded that on a microscopic level cardiac propagation is stochastic and not as uniform as it appears on a larger

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Introduction

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macroscopic scale. Thus, small input changes, may produce large changes in the propagation process, i.e. a change in the direction of propagation can evoke considerable changes in the gap junction delay and in the microscopic spread of excitation. Spach and Heidlage [1995] suggested this stochastic nature of propagation, a natural antiarrhythmic factor, by reestablishing the general feature of the wavefront after small variations. Depending on the direction of propagation, similar changes in Vmax have been observed in anisotropically grown cardiomyocyte cultures if major discontinuities existed using voltage-sensitive dyes [Fast and Kle´ber, 1994; Fast et al., 1996; Rohr, 1995]. In additional computer simulations these authors could show that the difference between longitudinal and transverse depended on the degree of anisotropy and the pattern of gap junctions. The difference was abolished in normally grown cell cultures. Since in the ageing heart or after myocardial infarction cells become separated by connective tissue layers, Fast et al. [1996] investigated the effect of longitudinal clefts on action potential propagation. They found that such structures result in local differences in Vmax and in the local action potential upstroke. In addition, they showed that lack of Cx43 led to local conduction block and to disturbance of the activation spreading. This may be of importance for the arrhythmogenesis in chronic heart diseases which are characterized by a reduction in Cx43 and changes in the Cx43 distribution pattern (see chapter 6). In a high-resolution mapping of the cardiac activation in isolated rabbit hearts, the stochastic nature of propagation mentioned above may be reflected by the beat-to-beat variability observed by others [Dhein et al., 1990]. Tachycardic arrhythmia are often maintained by reentrant circuits. Thus, the initiation of reentry is still a focus of arrhythmia research. It is, however, not in the scope of this chapter to give a detailed complete overview on the topic of arrhythmogenesis [readers interested in this are referred to the literature, e.g. Janse and Wit, 1989; Spach and Josephson, 1994]. This chapter focusses on the role of some biophysical properties of the tissue and the gap junctional intercellular communication. Different mechanisms have been discussed and demonstrated in various models. Intrinsic repolarization inhomogeneities as generated by two stimuli (extrasystoles) S1 and S2 at the same site can lead to reentry without the requirement of an anatomical obstacle [Moe et al., 1964]. As shown by Allessie et al. [1977], the circulating excitation waves can create a functional central obstacle in the form of a centrally nonexcited region. Following this concept, which is known as the ‘leading circle concept’, the membrane potential of these central fibers is held above threshold due to the electrotonic influence of the circulating wavefront. As a result, centripetal waves cannot shortcut the circuit and are extinguished in the center. For this type of reentry a minimum area of 30–50 mm2 is required. In 1966 Krinsky

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demonstrated in a 2-dimensional isotropic model that localized delays of conduction within the circuit reduce the minimum perimeter of the reverberator so that it may be less than the wavelength of refractoriness k (k>RP * V; RP>refractory period, V>average conduction velocity). This is substantially different from the classical reentry model requiring a central obstacle with the reentrant circuit path length equalling the wavelength of refractoriness (k>RP * V) [Wiener and Rosenbluth, 1946]. Similar forms of reentry can be initiated by S1 and S2 at different sites, but require considerable large areas [Davidenko et al., 1990; Van Capelle and Durrer, 1980]. Focusing on the passive electrical properties of the tissue, Spach et al. [1981] demonstrated that cardiac tissue is a nonuniform anisotropic medium. This has many implications for the theory of initiation of reentry and at least for the involvement of gap junctions. First of all, anisotropic reentry can occur in tissue without depolarization inhomogeneities. Second, microreentry is possible in small areas of =10–15 or even =2 mm2 [Spach et al., 1988]. This nonuniformity can be caused by either microfibrosis with connective tissue septae separating the fibers or by changes in the cellular coupling due to inhomogeneities in the distribution of gap junctions (see above), especially with sparse side-to-side coupling. This can lead to two different pathways: one of fast longitudinal conduction with a longer refractory period and another with a very slow conduction and shorter refractory period. As explained above the longitudinal conduction is more sensitive to premature stimuli, causing conduction failure in the longitudinal direction whereas transverse propagation is maintained. This situation can initiate microreentry as shown by Spach et al. [1988]. The vulnerable period of anisotropic reentry is confined to the interval between the refractory periods of longitudinal and transverse propagated action potentials [for review see Spach and Josephson, 1994]. Such mechanisms may also play an important role in atrioventricular (AV) reentry since the transitional zone of the AV node was found to exhibit markedly nonuniform anisotropic properties. What is the role of the gap junctions? By coupling the myocardial cells in both directions (longitudinal and transverse) they are responsible for the biophysical properties of the tissue. A reduction in gap junction distribution or a closure of the gap junction channels causes nonuniformities and discontinuities which alter the biophysical properties of the tissue and make it more prone to nonuniform anisotropic reentry. According to the model proposed by Krinsky [1966], a reduction in gap junctions or a closure of gap junction channels will lead to local slowing of conduction, thereby allowing smaller perimeters of reentrant arrhythmia. In addition, slowing of conduction is generally believed to be a risk factor for initiation of reentry. Since in many

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Introduction

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cardiac diseases, including acute (e.g. regional ischemia) and chronic (healed infarction) states, changes in cellular coupling have been observed (see chapter 6) , it is tempting to speculate that altered intercellular communication takes part in the formation of the arrhythmogenic substrate [Quan and Rudy, 1990; Joyner, 1982] by introducing discontinuities. In the following paragraphs several types of arrhythmia will be discussed with regard to the underlying mechanisms. Since it would be out of the scope of this book on gap junction channels to discuss all possible mechanisms of arrhythmia in detail, readers interested in a complete detailed review of the pathophysiology and clinics of arrhythmia are referred to the reviews by Janse and Wit [1989] and Pogwizd and Corr [1987, 1990] and to the specialized literature. Regional ischemia in the course of atherosclerotic coronary artery disease is one of the most important causes of arrhythmia in the Western industrial world. These arrhythmias start with or often degenerate into ventricular fibrillation and are the main cause of sudden cardiac death in these countries. However, in the course of ischemia and infarction the mechanisms by which arrhythmia is induced vary with the duration of ischemia. In the acute phase of ischemia, i.e. within the first 2–4 h ventricular arrhythmias often occur. Within the first 30 min of ischemia two types of arrhythmia can be distinguished: type-1a arrhythmias occur after 2–10 min with a peak at 5–6 min. They often originate from the subepicardium and the mechanism is assumed to be associated with diastolic bridging leading to reentrant arrhythmia. Besides this, non-reentrant type-1a arrhythmias can also occur which may be due to the flow of injury current across the ischemic border causing ectopic activity [Janse and Wit, 1989]. Type-1b arrhythmias occur later at 12–30 min with a peak between 15 and 20 min and are considered to be due to a partial recovery of dU/dtmax and the action potential duration following catecholamine release from the sympathetic nerve terminals. Apart from this, at the time of the occurrence of type-1b arrhythmia, gap junctional uncoupling with an increase in intercellular resistance has been described (for details see chapter 6) [for review of ischemia-related arrhythmia see, Janse and Wit, 1989]. The acute phase of ischemia is followed by 3–6 h of predominantly sinus rhythm. Thereafter, the number of ventricular ectopic beats increases. In the subacute phase of infarction (12–24 h) ventricular arrhythmias often occur. One of the mechanisms involved is reinfarction. If there is no acute reinfarction involved, these arrhythmias have been suggested to originate from surviving strands of Purkinje fibers in the subendocardium. The predominant mechanism has been postulated to be abnormal automaticity in these fibers. These fibers exhibit an increased sensitivity for catecholamines. In some cases a combination of focal activity and reentry in these fibers may be possible.

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The arrhythmias mainly observed are ventricular tachycardias, ventricular premature depolarizations and accelerated idioventricular rhythms as well as atrioventricular dissociation. During the process of healing the biophysical properties of the tissue change dramatically. The connexin 43 content is reduced especially in the healed and border zone (see chapter 6 for details) and the necrotic tissue is replaced by scars. This implies that the pathways of activation can change during this process. It has been found that during the chronic phase after myocardial infarction, ventricular arrhythmias and sudden cardiac death can occur. Programmed stimulation of the ventricles can induce both sustained and nonsustained ventricular tachycardia and fibrillation. Within the infarcted area islets of surviving myocardial cells may be found. These cells show somewhat altered electrophysiological properties with maximum diastolic potential, action potential amplitude and duration as well as maximum upstroke velocity being moderately reduced during the first week after the infarction. Thereafter, these parameters return to normal except the action potential duration. Conduction velocity transverse to the fiber axis is reduced, while velocity parallel to the fiber is nearly normal, which may reflect the loss of side-to-side connections and the incorporation of connective tissue (see chapter 6). The action potential characteristics of surviving cells return to normal at later times during the process of healing, i.e. after more than 1 month, whereas the irregularities in conduction persist and probably participate in forming the arrhythmogenic substrate. Reentrant arrhythmias have been observed during that phase due to epicardial reentry in the border zone and in some cases to intramural or subendocardial reentry [for a detailed review see, Janse and Wit, 1989]. Because of the complex structure of the AV node involving highly specialized interdigitating fibers with certain connexins forming the basis of intercellular communication (see chapter 3) it is tempting to speculate (although not shown experimentally with certainty) that changes in the distribution of gap junctions or alterations in the gap junction conductance may contribute to bradycardiac arrhythmia like AV conduction blocks or to tachycardic arrhythmia, e.g. AV reentry. Similarly, changes in the gap junction distribution or regulation of conductance may participate in sinuatrial block. However, experiments on this topic are still lacking. At least three types of disturbance in the intercellular communication have to be distinguished: (1) separation of the cardiac muscle fibers by strands of connective tissue as occurring in microfibrosis; (2) changes in the distribution of gap junction channels, and (3) changes in the conductance of gap junctional channels either by alteration of the open probability or of the single channel conductance.

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Introduction

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What is the role of gap junctions in the initiation and perpetuation of arrhythmia? Cole et al. [1988] compared experimental data with computer simulations and analyzed the relationship of gap junction uncoupling and discontinuous propagation in the heart. In summary, they found that reducing the gap junction coupling led to a decrease in propagation velocity as well as to an increase in Vmax and in the time constant of the action potential foot (sfoot). The phase-plane loops became nonlinear. Shaw and Rudy [1995] demonstrated in a cable model, using the Luo-Rudy membrane model, that the vulnerable window of unidirectional block depends on both membrane excitability and intercellular coupling. A uniform decrease (* 0.25) in intercellular coupling increased the time of the vulnerable window by a factor of 3.6, whereas a decrease in excitability (* 0.25) led to an increase by the factor 0.4. When inhomogeneities were present in the modelled fiber, the model became more sensitive to inhomogeneities in membrane excitability. As shown experimentally by Delmar et al. [1987] during impairment of the intercellular gap junctional communication (by 1.5 mmol/l heptanol) transverse propagation becomes more vulnerable to conduction block. Such a spatial dissociation in propagation can form the basis for the occurrence of reentry (see above). However, in the course of ischemia both conduction velocity in longitudinal and transverse direction are reduced (from 50 to 33 and from 21 to 13 cm/s, respectively), so that the ratio between longitudinal and transverse conduction was only slightly changed [Kle´ber et al., 1986] (from 2.38 to 2.54). These results demonstrate the changes associated with an alteration in the intercellular communication, which probably contributes to enhanced arrhythmogeneity in situations with a reduction in the number of gap junctions, e.g. ischemic heart disease (see chapter 6) [Peters, 1996], or reduced gap junction conductance, e.g. acute ischemia, hypoxia and acidosis (see chapters 4 and 6).

1

Introduction

12

2

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Structure and Diversity of Gap Junction Channels

The gap junctional channel has two main functions: (a) to allow transport of small molecules (MW=1,200–1,900 as established in Chironomus salivary gland gap junctions) [Schwarzmann et al., 1981; Simpson et al., 1977] such as intracellular messengers, small peptides and proteins, nucleotides as well as injected dyes as fluorescein or lucifer yellow, which are often used in dyecoupling studies (see chapter 8) from one cell to another thereby forming a syncytium, and (b) to provide electrical coupling between cells with or without rectifying properties thereby allowing the propagation of an action potential from one cell to another. Thus, the pore of the channel has to exhibit properties as a transcellular transport pipe and as an electrical connector which can be turned on and off. The structure of gap junction channels has been investigated employing electron microscopy, X-ray diffraction methods and molecular biology. By these techniques it was possible to define a model of the channel which is now widely accepted. According to these investigations a gap junctional channel is a polymeric structure consisting of 12 proteins called connexins. Corresponding to their molecular weight these connexins are designated as connexin 38 (Cx38; 38 kD), connexin 40 (Cx40; 40 kD), connexin 43 (Cx43; 43 kD) and so forth. The connexin family comprises at least 15 different isoforms (see fig. 8), among which the isoforms Cx37, Cx40, Cx43, Cx45 and Cx46 are expressed in mammalian cardiovascular tissue. The whole pipe-like channel is made of two connexons which are contributed by the two adjacent cells. Such a connexon begins at the cytoplasmic surface of the plasma membrane, crosses the lipid bilayer and ends up in the extracellular space between two adjacent cells. In the neighboring cell another connexon is connected up to this structure and both connexons then build up the gap junctional channel passing the cell membranes of the two adjacent cells. These hexameric connexons consist of 6 polypeptide subunits, the so-called connexins, which surround the inner core of the channel. These connexins have been intensively investigated in the last years so that today the amino acid sequences of a considerable number of them is clarified. A connexin has 4 transmembrane domains (M1, M2, M3, M4), 2 extracellular loops (E1, E2), 1 intracellular loop and the N terminus and C terminus both at the cytoplasmic side. The extracellular loops and membrane-spanning domains are highly conserved

13

[Caterall, 1988] comparing different connexins and different species, whereas the intracellular loop as well as the N terminus and C terminus exhibit a higher variability. Within the M3 segment typically phenylalanines are present and located next to the charged groups. This is probably an important feature allowing the twisting motion which is required for channel opening and closure. The N-terminal region exhibits about 50% identity (except for Cx30 and Cx32, which are rather closely related). The C terminus varies in length from 18 amino acids (Cx26) to 156 amino acids (Cx43) or even 191 amino acids (Cx46). In these cytoplasmic parts of the connexin the target sequences recognized by regulatory protein kinases (see chapter 4) can be found. The extracellular loops are thought to participate in the process forming a complete gap junctional channel from two hemichannels. In this context it is an interesting feature of both extracellular loops E1 and E2 that each contains three cystein residues spaced by either 6, 5 or 4 amino acids. These cysteins are found at the identical position in all connexins (Cx26, Cx30, Cx32, Cx38, Cx43). It has been speculated that they play a role in channel formation by disulfide bonds, although so far there are no hints on intermolecular disulfide bonds from SDS-PAGE studies determining the molecular weight of connexins. In contrary, in Cx43 channels it was shown that the connexon integrity is maintained by noncovalent bonds and that there are no intermolecular disulfide bonds [John and Revel, 1991]. Only the possibility of intramolecular disulfide bonds has been suggested [Dupont et al., 1989; John and Revel, 1991]. The forming process of the channel is not yet well understood. The most abundant gap junction protein found in the heart is Cx43. After the liver gap junction protein (Cx32) had been cloned by Paul [1986], Beyer et al. [1987] looked for cardiac mRNA from rat hearts which would crosshybridize with the cDNA for rat liver Cx32. They were able to identify three cDNAs together spanning 2,768 base pairs with a single open reading frame of 1,146 base pairs coding 382 amino acids with a calculated molecular mass of 43,036 kD. The isolated rat cDNA sequence has a first initiation codon (ATG) at base 202 followed by the 1,146-base pair reading frame and a termination codon at base 1,348. The coding region is followed by 1,218 bases of the 3-untranslated sequence which includes several termination codons but lacks a polyadenylic acid tail. The predicted molecular mass fits well with that obtained from biochemical isolation of cardiac gap junction proteins by Manjunath and Page [1985, 1986] using SDS-PAGE (44–47 kD). The difference may be due to uncertainties with the SDS-PAGE method or to a co- or posttranslational phosphorylation as is suggested in the study by Crow et al. [1990]. Partial amino acid sequencing studies of the isolated protein revealed considerably high homology between the found and predicted sequence near the N terminus [Manjunath et al., 1987; Nicholson et al., 1985]. From the sequencing studies it has been

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Fig. 1. Amino acid sequence of chicken Cx43 according to Veenstra et al. [1993]. Possible targets for phosphorylating enzymes are underlined.

concluded that the first methionine residue is removed posttranslationally so that in the mature protein the first amino acid is a glycine. Regarding Cx43, Beyer et al. [1987] predicted a pI at 10.19 indicating a very basic protein. According to this study Cx43 has 34.3% polar and 42.4 nonpolar amino acids, 9.4% acidic and 13.9% basic residues at neutral pH. Because of the 53 basic amino acids which include 8 histidine residues facing 36 acidic residues, a net positive charge of 17 would result. Within the molecule 4 hydrophobic regions have been identified in alternation with hydrophilic regions using the hydropathicity plot of Kyte and Doolittle [1982]. From these data the 4 membrane-spanning domains were predicted and the structure for Cx43 as given in figure 1. According to Lau et al. (1996) and Delmar et al., (1995) phosphorylation may occur at the following sites: at the serine residues 364, 368, 372 (R-X-S motif for PKA, PKG and PKC), 296, 365, 369, 373 (R-X-X-S motif for PKA, PKG, PKC and CaMK II), 244, 306 (K-X-X-S motif for PKG, PKC), 364, 368, 373 (R-X-S-X-R motif for PKC), 297, 364, 368, 372 (S-X-R motif for PKG, PKC), 262 (S-X-K motif for PKG, PKC and at threonine residue 290 (K-X-X-T motif for PKG, PKC) at tyrosine residue 265 (vSRC tyrosine kinase)

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Fig. 2. Phase contrast microscopy of rabbit cardiac muscle. Note the ‘Glanzstreifen’ with the intercalated disk (arrow). ¶1,000.

as well as at the serine residues 279, 282 and 255 which can be phosphorylated by MAP kinase. The numbers refer to rat Cx43 as given here (membrane domains 1-4 are given in italics): 1 51 101 151 201 251 301 351

MGDWSALGKLLDKVQAYSTAGGK VWLSVLFIFRILLLGTAV ESAWGDEQS AFRCNTQQPGCENVCYDKSFPISHVR FWVLQIIFVSVPTLLYLA HVFYVM RKEEKLNKKEEELKVAQTDGVNVEMHLKQIEIKKFKYGIEEHGKVKMRGG LLRTYIISILFKSVFEVA FLLIQWYIYGFSLSAVYTCKRDPCPHQVDCFL SRPTEKT IFIIFMLVVSLVSLALNI IELFYVFFKGVKDRVKGRSDPYHAT TGPLSPSKDCGSPKYAYFNGCSSPTAPLSPMSPPGYKLVTGDRNNSSCRN YNKQASEQNWANYSAEQNRMGQAGSTISNSHAQPFDFPDDNQNAKKVAAG HELQPLAIVDQRPSSRASSRASSRPRPDDLEI

The spatial structure of the channel has been investigated for a long time. In the beginning, light microscopists described intercalated disks which appeared as bands transverse to the longitudinal axis of the cardiac muscle fiber [Eberth, 1866]. With modern phase contrast microscopes they can easily be seen as shown in figure 2. These bands were a matter of discussion for a long time until in 1954, for the first time, Sjo¨strand and Andersson used electron microscopy to investigate intercalated disks in ultrathin osmium tetroxide-fixed sections of the mouse heart revealing that the disks were indeed transverse cell boundaries. Subsequently, several investigators reproduced their finding [Lindner, 1957; Moore

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and Ruska, 1957; Poche and Lindner, 1955] and some new methods were used in the investigation of these cell boundaries. It became obvious that the intercalated disk contains three distinct structures: fascia adherens (the main portion of the disk); macula adherens or desmosome, and nexus. The fascia adherens is made of two parallel lipid bilayers separated by a distance of ˚ , whereas the desmosome is a more complex and almost laminated 200–300 A structure built from the two adjacent membranes. The nexus is the zone of close contact between the cells containing the gap junction channels. Our present image of a gap junction channel is based on the X-ray diffraction studies of Makowsky [1988], Makowsky et al. [1977, 1984] and Tibitts et al. [1990] and the low irradiation electron microscopy [Gogol and Unwin, 1988; Sikewar and Unwin, 1988; Sosinsky et al., 1988] as well as the use of antibodies directed against specific amino acid sequences in the subunits in order to get information on the topology [Milks et al., 1988; Zimmer et al., 1987] and the cloning of cDNA [Kumar and Gilula, 1986; Paul, 1986]. From these studies the hexameric character of the channel became evident and, as already mentioned, 6 connexins together form a connexon with an inner pore. Via the extracellular loops 2 connexons are interconnected to each other thereby constituting the full gap junction channel. This structure has been subject to intensive X-ray diffraction studies and electron microscopy. These studies revealed the spatial structure of the channel with a total length of ˚ and about 52 A ˚ in the portion within the lipid bilayer approximately 100–150 A of each side [Chen et al., 1989] and the two parallel membranes separated by ˚ gap spanned by the channels subunits [Beyer et al., 1995]. The diameter a 20 A ˚ and about 15 A ˚ in the extracellular of the channel ranges between 20 and 30 A half of the lipid bilayer. The inner pore appeared solvent filled, and is =2.5 nm at its widest point [Severs, 1994a, b]. Transmission electron microscopy of positively stained cross-sectioned cardiac gap junctions of mammalian ventricles or atria revealed a 7-layered structure: two 3-layered lipid membranes and the gap between them. More recently Perkins et al. [1997] developed a ˚ height and 6 lobes prothree-dimensional model of the connexon with 50 A truding from the extracellular surface, that would dock with an opposing connexon to form an intercellular channel. Using the freeze fracture technique, electron microscopy and laser scanning confocal microscopy, it became obvious that these gap junctional channels are arranged as a cluster of channels with about 50 channels within one disk as stated by Gourdie et al. [1990]. From these studies and results Makowsky et al. [1977] developed a threedimensional model for the channel. According to these studies the gap junction channels are arranged in clusters as shown in figure 3. A model of a single channel is given in figure 4.

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Fig. 3. Drawing of a cluster of gap junction channels.

Fig. 4. Model of a single gap junction channel and a connexon.

The next question to answer was the mechanism of closure of the channel. It is widely accepted now that the channel is closed by a rotational movement of the hexamer [Unwin and Ennis, 1984; Unwin and Zampighi, 1980] as illustrated in figure 5. This twisting motion closing the central channel is possible since the a-helix of the connexins, which is the part located within the lipid bilayer, is inclined with respect to the axis of the whole connexon [Milks et al., 1988].

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Fig. 5. Model of gap junction channel opening and closure by a slight twisting motion of the connexon (bent arrow) which opens the central channel. Note the inclination of the a-helical segments of the connexins with regard to the axis of the whole connexon as proposed by Unwin and Zampighi [1980].

The next issue to discuss is the diversity of connexins, i.e. the various isoforms, and species variability. Gap junctional channels exist in a broad variety of tissues including the heart, vascular system, brain, epithelial tissues, uterus, lens cells, pancreas and kidney. However, these tissues are connected by different isoforms of gap junctional connexins which can be distinguished with regard to their molecular weight. These differences are mainly due to various lengths of the C-terminal loop. The smallest connexin is Cx26 with an approximate molecular weight of 21–26 kD as determined using SDS-PAGE. The C terminus consists of 18 amino acids. The N-terminal region contains 22 amino acids. Rat Cx26 has been found in liver hepatocytes [Traub et al., 1989; Zhang and Nicholson, 1989], pinealocytes, leptomengineal cells [Dermietzel et al., 1989], pancreatic acinar cells [Traub et al., 1989], endometrium [Risek et al., 1990] and in various other tissues including lung, kidney, spleen, intestine, stomach and testes [Zhang and Nicholson, 1989]. The amino acid sequence is given in figure 6. A connexin with a molecular weight of 30 kD (Cx30) has been isolated and cloned from xenopus liver and was also found in the lung, intestine, stomach and kidney of xenopus [Gimlich et al., 1988]. The C terminus is enlarged to 58 amino acids. The N terminus contains 22 amino acids. The amino acid sequence is also given in figure 6. Another connexin with a molecular weight of 32 kD, Cx32, was cloned from human liver [Kumar and Gilula, 1986], rat liver [Paul, 1986] and was also found in hepatocytes [Paul, 1986; Traub et al., 1989], stomach, brain and kidney [Paul, 1986] as well as in pancreatic acinar cells [Dermietzel et al.,

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Fig. 6. Amino acid sequence of Cx26, Cx30 and Cx32. Amino acids are given by a one-letter code [according to Paul et al., 1986; Bennett et al., 1991; Gimlich et al., 1988].

1984] and oligodendrocytes [Dermietzel et al., 1989]. The C terminus consists of 76 amino acids and the N terminus of 22 amino acids. The amino acid sequence is given in figure 6. The second group of connexins, including Cx37, Cx38, Cx40, Cx43 and Cx46, is characterized by a longer N terminus which is elongated by 1 amino acid in position 3. It is believed that these two groups of connexins represent two parts of a phylogenetic tree of the connexin family as pointed out by Bennett et al. [1991] (see also fig. 8). The first protein of this second group is Cx38 which has been cloned from xenopus oocytes and was also found in the embryo [Ebihara et al., 1989; Gimlich et al., 1990]. The C terminus consists of 120 amino acids. It has a high homology to mouse Cx37. Studies in a xenopus oocyte expression system revealed that Cx38 by itself exhibits only poor channel forming ability, but is highly effective in forming hybrid channels with Cx43. Thus, it has been suggested that the function of Cx38 is to form hybrid rather than symmetrical channels [Werner et al., 1993]. The full amino acid sequence of Cx37 is given in figure 7. Cx37 is an isoform belonging to the same branch of the Cx family

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Fig. 7. Amino acid sequence of Cx37, Cx40, Cx43 and Cx45. Amino acids are given by a one-letter code [according to Kanter et al., 1992; Reed et al., 1993; Fishman et al., 1990]. Note that the N terminus is enlarged by one amino acid in position 3 as compared to Cx26, Cx30 and Cx32.

tree, and has been found in rodents (mice, rats) and is highly expressed in lung [Willecke et al. 1991]. One of the last connexins which has been characterized is Cx40 [Kanter et al., 1992], which is one of the connexins typically found in the heart. However, it is preferentially expressed in the lung [Hennemann et al., 1992b]. Within the heart it has been discovered in atria (human, but not in all species, e.g. rat atria does not express Cx40), in the conduction system and in the vascular endothelium [Bastide et al., 1993]. Chick Cx42 has been considered to be the homologue of mammalian Cx40 with 70% of the amino acids being identical [Kanter et al., 1992]. Thus, it is referred to as Cx40. The ratio between Cx40, Cx43 and Cx45 in heart can be altered in the course of cardiac diseases (for a detailed discussion see chapter 6). The full amino acid sequence is given in figure 7.

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Table 1. Residue

Human

Rat

Xenopus

124 16 234 251 253 257 263 344 347

D I K S A A Q S L

E V R T P S P A V

D V K N A G P M1 G1

1

For Xenopus the residue number for these amino acids is smaller by 3.

Cx43 is the gap junction protein most abundantly found in the hearts of various species including human, dog, chicken and rat. The molecular weight as determined by SDS-PAGE ranges from 42 to 45 kD. Besides in the heart, Cx43 has been found in uterine muscle, granulosa cells, smooth muscle cells, kidney, eye (cornea and lens), epithelium [Beyer et al., 1987, 1989; Risek et al., 1990], liver, spleen, ovary [Gimlich et al., 1990], in fibroblasts [Musil et al., 1990a], in astrocytes and leptomeningeal cells [Dermietzel et al., 1989; Yamamoto et al., 1990]. mRNA for Cx43 has been found in endothelial cells, pericytes and vascular smooth muscle cells [Larson et al., 1990]. Cx43 seems to be highly conserved between the species. A homology of 92% between chick Cx43 and rat Cx43 has been found [Musil et al., 1990a]. Its N terminus exhibits 23 amino acids and its C-terminal loop 156 amino acids. The full amino acid sequence is given in figure 7. Using a rat Cx43 probe and a 10-day chick embryo cDNA library, chick Cx42 (see above) and chick Cx45 were identified [Beyer, 1990]. This connexin is developmentally regulated with higher levels of its mRNA in early embryos than in more mature organisms [Beyer, 1990; Veenstra et al., 1993]. In the extracellular loops E1 and E2 the typical three cystein residues can be found. Mouse Cx45 consists of 396 amino acids and has a molecular weight of 45, 671 [Willecke et al., 1993]. There is a homology of 85% between chick Cx45 and canine Cx45 [Kanter et al., 1992]. The amino acid sequence is given in figure 7. Finally, the longest connexin is Cx46, which is also expressed in rat heart and has been cloned from lens [Beyer et al., 1988]. This connexin exhibits the

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Fig. 8. The phylogenetic ‘connexin family tree’ according to Bennett et al. [1995]. Branching points with closed ovals represent gene duplications whereas branching points without ovals represent speciation. X>xenopus; Ch>chicken; ms>mouse; r>rat; bov> bovine; c>canine; h>human.

longest C terminus known so far with 191 amino acids, the total protein consisting of 416 amino acids [Paul et al., 1991]. In addition, an eye lens cell protein known before as MP 70 has recently been identified as Cx50 [White et al., 1992]. Regarding species variability there are some points to mention. First, a variability in the distribution pattern of a distinct connexin isoform is possible, for example with Cx40 which is normally found in atria of many species but not in rat atria. The details for these species differences with regard to the distribution of connexins in the heart are given in chapter 3. Second, the amino acid sequence can be altered and it has been shown that there are indeed some single amino acids which vary depending on the species. In Cx43 the following differences are reported [for review see, Bennett et al., 1991] (table 1). In some cases such variability has consequences for the regulation of the gap junction channels. Thus, in rat Cx32 the serine residue at position 233 is

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phosphorylated by a cAMP-dependent protein kinase, but this is unique to rCx32. Finally, a phylogenetic analysis of the connexin family has been carried out restricting the analysis to the two major conserved regions in connexin genes, and a ‘family tree’ was proposed (fig. 8). Since the distance between amphibian and mammalian orthologues is considerably smaller than between group I and II, it was suggested that the branching between the two groups occurred rather early in phylogenesis, i.e. in the early or even before vertebrate divergence. The two extra cellular loops E1 and E2 are the most conserved regions of the connexins with three invariant cysteins (figures 6 and 7). The transmembrane domains M1-M4 are somewhat less well conserved, while the cytoplasmic loop and the C-terminal are the most variant regions of the molecule.

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3

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Distribution of Gap Junctions in the Heart

In the first part of this chapter the distribution of gap junctions within a cell is discussed. In the second part the connexin pattern in the heart is described, and in the third part the expression of various connexins in the vasculature is outlined. Heart muscle fibers are coupled by gap junctions. These intercellular channels provide the exchange of small molecules (=1,000 D), like second messengers, between the cells and they allow electrical coupling. Thus, these cells connected to each other form a syncytium. However, from mapping studies it became evident that under certain conditions, e.g. regional ischemia, the ischemic region uncouples. In addition, mapping studies demonstrated that there is a special activation pattern which accounts for a directed activation of the whole heart. This activation pattern exhibits a considerable similarity from beat to beat. It is well known that the conduction velocity varies between 0.3 and 0.6 m/s in the ventricles and 1.0 m/s in the Purkinje system. On the other hand conduction is delayed in the AV node. In addition, the activation has to be transduced from the sinuatrial node to the atria, and from the endings of the Purkinje fibers to the ventricular myocytes. Thus, the coupling within the tissue and between various cells becomes an important feature to provide the normal impulse conduction. From the above-mentioned considerations an association of the different functions and demands with different types of coupling can be concluded. Thus, in general, Cx40 can be found in the conduction system whereas ventricular myocytes are coupled by Cx43. In this chapter, the distribution of the various connexins within the cardiac tissue will be described. First, the distribution of the gap junction channels within a cell will be outlined. In the foregoing chapter, it was found that the intercalated disks seen on light microscopy contain the gap junction channels. However, it remains uncertain how the gap junction channels are distributed within a disk or how the cell-to-cell boundary is shaped. This question has been resolved using the freeze-fracture technique supplemented with image-processing systems. Briefly, to freeze-fracture cardiac muscle specimens, these specimens have to be fixed with glutaraldehyde in order to crosslink the proteins within the tissue, and incubated with glycerol in order to prevent ice crystal formation in the course of the freezing procedure. After freezing and fracturing in vacuum the specimens have to be unidirectionally stained in vacuo by exposure to platinum

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Fig. 9. Distribution of the gap junctions, desmosomes and fascia adherens in an intercalated disk of a cardiomyocyte as assessed by electron microscopy of freeze-fractured rat and rabbit hearts according to Severs [1990].

and carbon vapor at an angle of 45º, followed by deposition of carbon at an angle of 90º. Details of the method and variations in this technique are described in the book edited by Rash and Hudson [1979] and the application for gap junction research in the review by Severs [1989]. Using this technique [Severs, 1990] and others (transmission electron microscopy of positively stained serial ultrathin sections and scanning electron microscopy) [Hoyt et al., 1989] helped to clarify the arrangement of the gap junctions within the intercalated disk. It became obvious that the transverse cell boundary is not a plane disk but consists of several processes which interdigitate with the corresponding processes of the adjacent cell. These interdigitating membranes were formerly described as the ‘plicate segment’. Within this plicate segment the gap junctions were found to be located in the finger-like processes and the interface between the myocytes parallel to the fiber axis as shown in figure 9. From figure 9 it becomes clear that the fascia adherens is located transverse to the fiber axis on the cell processes and at the side walls of these processes gap junctions are located in clusters and desmosomes. According to Hoyt et al. [1989] the gap junctions are arranged in a more or less ribbon-like fashion

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whereas according to the model of Severs the gap junctions are distributed over a wider range. From a biophysical point of view this makes an important difference because the electrical transfer from one cell to another will be influenced by this structural arrangement. This would affect the spatial distribution of current flowing from one cell to another and could possibly affect the efficacy of coupling. However, presently it is not absolutely certain whether the model of Hoyt et al. [1989] or of Severs [1990] is correct. Hoyt et al. [1989] found that about 3.6 myocytes overlap so that each is connected to 9 other myocytes. The relatively new technique of laser scanning confocal microscopy enabled the determination of the number of gap junctions within one disk to be in the order of 50 or even more [Gourdie et al., 1990] with a diameter of up to 1.3 lm/gap junction. Within the gap junction the channels themselves are arranged as parallel pipe-like structures. From freeze-fractured junctions it has been estimated that about 12.9 · 103 channels are located in 1 lm2 gap junction (rat right ventricular myocardium) [Chen et al., 1989]. The surface of the intercalated disk is occupied to 5.7×0.6% by gap junctions (canine atrium) [Spira, 1971], 3.3% (right bundle, calf ) [Arluk and Rhodin, 1974], or even 12.7–15.1% in canine left ventricular subepicardial myocardium [Hoyt et al., 1989]. The rest of the intercalated disk is made of fascia adherens and desmosomes. In the crista terminalis of the canine heart the gap junction profile length has been estimated to be in the order of 3.2–3.8 lm/100 lm intercalated disk length with 11–12 gap junctions/100 lm intercalated disk length and a mean gap junction profile length of about 0.3 lm [Saffitz et al., 1994]. However, connexins do not seem to be restricted to the transverse cell boundaries, since they have also been detected in several specimens at the lateral cell side. For example Oosthoek et al. [1993b] demonstrated Cx43positive staining at the lateral cell side of human and bovine hearts (ventricles). Figure 10 shows another example from the rabbit heart, using an anti-Cx43 monoclonal antibody in cryostat sections of the rabbit left ventricle. Please note the distribution of Cx43 positivity at the transverse cell boundaries and at the lateral cell sides. In cardiac tissue mRNA for Cx37, Cx40, Cx43, Cx45 and Cx46 has been detected in dog, mouse and rat heart [Haefliger et al., 1990; Hennemann et al., 1992a, b; Kanter et al., 1992; Paul et al., 1991; Willecke et al., 1991]. However, in the heart, Cx40 and low levels Cx45, Cx43 is found most abundantly. The distribution of the various connexins exhibits a specific pattern with some species variability which will be discussed in the next section. Cx43 has been detected in many areas of the heart; however, only very low levels were found in AV node and sinus node. Cx43 was absent in AV

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Fig. 10. Photomicrograph demonstrating immunohistochemical staining for Cx43 in cryostat sections of the left ventricle of the rat heart. Objective: ¶40. neofluor achroplan, Zeiss. Magnification:¶400.

bundle and the bundle branches [Gourdie et al., 1992; van Kempen et al., 1991]. However, there are some discrepancies in the newer literature regarding the Cx43 distribution pattern: Bastide et al. [1993] could not detect Cx43 in the atrioventricular bundle and bundle branches of the rat heart. Similarly, Oosthoek et al. [1993a, b] found expression of Cx43 in the AV bundle and bundle branches in bovine and human hearts but a lack of Cx43 expression in the AV node and the center of the sinoatrial node of human and bovine heart. In the central sinus node of the rat heart these authors did not find Cx43 staining either. In contrast, Anumonwo et al. [1992] reported Cx43 in sinoatrial nodal cell pairs isolated from the rabbit heart, and Trabka-Janik et al. [1994] in the hamster sinoatrial node cells. However, these authors did not double stain the cells with an anti-a-smooth muscle actin antibody which is known to specifically stain sinoatrial node cells. Thus, it might be possible that the Cx43-positive cells were obtained from the border zone of the sinoatrial node. If Cx43 is investigated in the conduction system, it has to be taken into account that Cx43 is expressed in these tissues [van Kempen et al., 1995] only after birth.

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Cx40 was found in sinus node cells, atrium, AV node, AV bundle and bundle branches and Purkinje fibers. Cx45 was expressed at low levels in Purkinje fibers and ventricles of the canine heart [Kanter et al., 1993a, b, c]. Following the anatomy of the heart more systematically, in the sinoatrial node a center zone has to be distinguished from the periphery. It was found in the human and bovine heart that in the center zone no Cx43 was expressed, but that from this center wings extended toward the superior caval vein and toward the atrium. Whereas the center zone of the sinoatrial node was composed of cells, small myocytes, negative for Cx43, the cell size was gradually increasing within these wings. These wings interdigitate with strands from the atrium which were positive for Cx43. Nodal cells (negative for Cx43) were separated by strands of connective tissue from the Cx43 positive cells [Oosthoek et al., 1993b]. Similarly, ten Velde et al. [1995] described an abrupt change from negative staining for Cx43 in the sinoatrial node (guinea pig) to positive staining in the atrium. Cx43-negative strands from the sinoatrial node toward the crista terminalis became smaller in size and alternated with Cx43-positive layers becoming progressively broader in the direction of the crista. Lateral contacts between the Cx43 and the a-smooth muscle actin-positive sinoatrial node cells were found to be rather sparse. Thus, the authors concluded that the primary pacemaker seemed to be shielded from the hyperpolarizing influence of the atrium (which has a more negative resting membrane potential than the node) by gradually coupling due to geometric factors (interdigitating fibers) and not by a gradient in Cx43 density. In addition, these authors found that endocardial strands at the crista terminalis side of the sinoatrial node were Cx40 positive, but the node itself was negative. However, in canine heart Cx40 and Cx45 have been detected [Davis et al., 1994; Kanter et al., 1993c]. In the atrium gap junctions with Cx43 have been found immunohistochemically [Gros et al., 1994] in rat and guinea-pig hearts. Besides Cx43, Cx40 is also expressed in the atrium of several species including guinea pig [Gros et al., 1994], goat [van der Velden et al., 1996], dog crista terminalis [Saffitz et al., 1994], man [Davis et al., 1995], but not or only in some cases in the rat [Gros et al., 1994]. In human atrium moderate amounts of Cx40, Cx43 and Cx45 were determined [Davis et al., 1995]. The AV node is a highly specialized structure of the conduction system, which is designed for delayed conduction (with a rate-dependent delay) of the action potentials from the atrium to the ventricles. Thus, it as been hypothesized that in the AV node other gap junction proteins may occur than in the ventricular myocardium. Indeed, only low expression of Cx43 has been observed in rat AV-nodal tissue [Gourdie et al., 1992; van Kempen et al., 1991]. In accordance with this finding, Oosthoek et al. [1993a] could not detect Cx43 expression in human or bovine AV-nodal tissues. However, besides Cx40 Davis

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et al. [1995] did find some Cx43 and Cx45 in human AV node. The reason for this discrepancy is not yet known. Since, however, van Kempen et al. [1995] could show that the connexin distribution patterns are to a large extent comparable between various mammalian species, these data taken together show that Cx40 is probably the predominant connexin in the AV node, and Cx43 plays only a minor role. Cells of the AV bundle also express Cx40 and are lacking in Cx43 (rat heart) [Bastide et al., 1993; Gros et al., 1994]. However, this seems to depend on the species investigated. Thus, in guinea-pig hearts only Cx43 but not Cx40 could be detected in the AV bundle [Gros et al., 1994]. In the human heart Cx43 was found at the end-to-end intercalated disks of the AV bundle [Oosthoek et al., 1993a]. A similar pattern is seen in the bundle branches which exhibit high amount of Cx40, Cx43 and Cx45 in the human heart according to Davis et al. [1995]. On the other hand, in rat bundle branches Cx43 is absent [Gourdie et al., 1992; Gros et al., 1994; van Kempen et al., 1991], whereas in the guinea pig Cx43 immunoreactivity was detected but not Cx40 expression [Gros et al., 1994]. Regarding the Purkinje fibers cells are connected via Cx43 and Cx40. A threefold higher expression of Cx40 mRNA as compared to Cx43 mRNA occurs in the canine Purkinje fibers [Kanter et al., 1993b, c]. Only very low levels of Cx45 were found in this study in Northern blots. The immunostaining intensity corresponded to these findings. A slightly enhanced amount of Cx43 mRNA in Purkinje fibers as compared to ventricular muscle has been demonstrated in the study mentioned above. This difference in connexin distribution and density may contribute to the well-known differences in conduction properties [Purkinje fiber conduction velocity: up to 2–3 versus 0.3–0.4 m/s in ventricles). In contrast, in the adult rat heart no Cx43 was observed in the proximal Purkinje system [van Kempen et al., 1991], whereas Gourdie et al. [1992] did observe Cx43 in the Purkinje fibers of the rat. Gros et al. [1994] also stated that gap junctions of rat Purkinje fibers contain Cx43 and Cx40. In the human and bovine heart Cx43 is expressed in Purkinje fibers. In the bovine heart Oosthoek et al. [1993a] found a characteristic distribution pattern of Cx43 with positive staining along the entire plasma membrane facing other Purkinje fibers but not those facing connective tissue. This characteristic pattern was not seen in the human heart. In the ventricular myocardium Cx40, Cx43 and Cx45 have been detected [Kanter et al., 1994; Verheule et al., 1997] but only Cx43 and Cx45 have been found in the human heart in considerable amounts, whereas only a very low expression of Cx40 was observed which was located at the subendocardium and at endothelial layers [Davis et al., 1995].

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In general, in various parts of the conduction system higher amounts of connexin were found especially in the fast conducting tissues as compared to ventricular myocardium. Only minimal expression of Cx37 and Cx46 between occasional atrial and ventricular myocytes has been observed [Davis et al., 1995]. Besides the myocardium and conduction system, connexins are also expressed in the coronary vasculature [Christ et al., 1996]. The arterial smooth muscles behave like a syncytium and up- or downstream conduction of a response has also been seen in the endothelial layer. In addition, bidirectional signalling is required for regulation of the vascular tone, i.e. signal transduction from the perivascular nerves (and nerve varicositites) toward the vessel lumen and vice versa. Thus, it could be assumed that the cells of the vascular wall may be interconnected by some type of cell-to-cell contacts. Indeed, in vascular tissue three connexins are expressed: Cx37, Cx40 and Cx43. As an example, Cx40 was identified in the endothelial layer of cardiac blood vessels [Bastide et al., 1993], but was lacking in the smooth muscle cell layer of the arterial walls as was characterized in frozen sections stained for immunohistology. However, immunohistologically Cx43 was detected in smooth muscle cells of the media of pig coronary arteries as a discrete punctuation. Further investigation by transmission electron microscopy revealed a lack of the typical gap junctions in this tissue, although dye coupling between the smooth muscle cells was observed [Beńy and Connat, 1992]. The authors concluded that these arterial smooth muscles cells were coupled through isolated gap junction channels, and not, as in the myocardium, through clusters of channels which can be detected microscopically. However, in other vasculatures gap junctions between smooth muscle cells could be identified: junctional plaques were seen in the human corpus cavernosum and in the rat aorta with diameters of 0.2–0.5 lm [Campos de Carvalho et al., 1993; Christ et al., 1993], as well as in rat and hamster resistance arteries [Little et al., 1995]. Besides Cx43, Cx40 was also found. The distribution of Cx43 in a coronary vessel and a schematic diagram of the distribution of various connexins in the vessel wall are shown in figures 11 and 12, respectively. Furthermore, it was found that Cx43 is expressed more extensively in synthetic phenotype cells but only a few gap junctions were observed between contractile cells [Rennick et al., 1993]. Thus, gap junction formation and Cx43 expression may depend on the phenotype of smooth muscle cells. This may also account for the differences observed by different investigators. Gap junctions between endothelial cells in the vascular wall contain channels formed by Cx40, Cx43 and, in contrast to the media, by Cx37 (rat aorta) [for review see, Christ et al., 1996] with a reduced Cx43 density as compared to smooth muscle cells.

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Fig. 11. Immunohistochemical localization of Cx43 in a rabbit coronary arteriolar vessel. The lower figure gives the corresponding phase contrast microphotograph.

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Fig. 12. Drawing of the distribution of various connexin isoforms within the vascular wall.

Presently it is not certain whether gap junctions between endothelial and smooth muscle cells exist in the vascular wall. Such myoendothelial interconnections could be an interesting mechanism of signal transduction from the lumen or the endothelium to the media and might also contribute to upstream regulation of vascular tone, but, although theoretically anticipated, they have not been shown unequivocally until now. However, there is some ultrastructural evidence for myoendothelial gap junctions between endothelial cells and processes of the smooth muscle cells passing through fenestrae of the elastica interna [Beńy and Pacicca, 1994]. In addition, myocytes and fibroblasts can form functional gap junction channels [Goshima, 1970] which has been experimentally investigated in gap junctions formed from both cells cultured from neonatal rat hearts [Rook et al., 1989]. It was found that the conductance between myocytes was in the order of 43 pS, between fibroblasts about 22 pS and between myocytes and fibroblasts in the range of 29 pS, indicating that a heterojunction may exist between both cell lines. Such heterojunctions are presently one of the main interests in gap junction research, since many physiological phenomena regarding crosstalk between various tissues and developmental phenomena may be involved.

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An interesting phenomenon discovered by several investigators in recent studies is the occurrence of coexpression of various connexins in the gap junctions of one cell. Using double-label immunofluorescence on disaggregated canine ventricular myocytes, Kanter et al. [1993c] could demonstrate with laser scanning confocal microscopy and electron microscopy that cardiac myocyte gap junctions contain multiple channel proteins with Cx40/Cx43 and Cx43/Cx45 colocalization, i.e. an individual cell contains more than one connexin isoform. There are two principally different possibilities for coexpression: (a) a single hemichannel could contain multiple connexins, or (b) hemichannels consisting of only one isoform may join another hemichannel made of another isoform thereby constituting a heteromeric gap junction channel. Presently, it is not clear whether hemichannels of mixed composition naturally occur. However, pairs of Cx32/Cx43 have been shown to be functional [Swenson et al., 1989; Werner et al., 1989]. In addition, using polyclonal anti-Cx43 and anti-Cx40 antibodies in frozen sections of the rat heart, coexpression of Cx43 and Cx40 in ventricular myocytes was seen immunohistologically [Bastide et al., 1993]. However, it is uncertain whether Cx43 and Cx40 form heterotypic channels in the heart, since in injected oocytes they only form homotypic channels [Bruzzone et al., 1993]. Such heterotypic channels would be expected to possess a single channel conductance of about 50 pS and should exhibit a sensitivity to transjunctional voltage, since channels formed by Cx40 exhibit a single channel conductance of 86–236 pS and a great sensitivity to transjunctional voltage and those made from Cx43 28–67 pS (being rather insensitive to transjunctional voltage) and Cx45 about 29 pS as characterized in embryonic chick heart cells [Veenstra et al., 1992]. A colocalization of Cx43 and Cx40 has also been observed in the intercalated disks of the guinea pig atrium by Gros et al. [1994]. In addition, it was shown that Cx40, but not Cx43, can form heterotypic channels with Cx37 (Bruzzone et al., 1993). It can be suggested, that, if adjacent cells express incompatible connexins, this provides a mechanism for the formation of different compartments.

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4

...........................

Function and Physiology of Gap Junction Channels

In this chapter insight into the regulation and electrophysiology of gap junctions will be given and the current-voltage relationships for various connexins will be described. In the first part a special focus is on the physiological regulation of gap junctional opening and closure by calcium, sodium, magnesium, cAMP, ATP, pH, pCO2, leukotrienes, catecholamines and acetylcholine. In the second part of this chapter the functions of gap junctions in the heart and in the various regions of the heart and vasculature (endothelium and smooth muscle) are detailed. In the third part of the chapter electrophysiology and biophysics will be discussed. Thus, current-voltage relationships, single channel conductances, differences between findings with double-cell voltageclamp and with the dye-coupling method will be pointed out.

4.1 Regulation of the Channels Gap junctional channels, like many other ion channels, can be modulated via second messengers and via phosphorylation processes. Besides these, intracellular calcium and pH have been proven to be important regulators of channel function. In this chapter the short-term regulatory processes are considered, i.e. processes on a time scale of minutes. Besides this, regulatory processes are known which take place over a period of 30 min up to several hours and which involve formation or synthesis of new gap junction channels. The latter processes are described in the following chapter. Gap junction conductance (gj) of neonatal rat heart cells varies with temperature (37 ºC, 48.3 nS; 14 ºC, 21.4 nS; –2 ºC, 17.5 nS) [Bukauskas and Weingart, 1993] so that gj has been assumed to be at least in part enzymatically controlled. Several protein kinases are known to be involved in the regulation of the gap junction channels. However, the situation is rather complicated since the same protein kinase may enhance or reduce gap junctional conductance in different tissues or in different species. Thus, generalizations should be avoided and the specific condition has to be taken into account. One of the first to be described was protein kinase A (PKA), the cAMP-dependent protein kinase, which can enhance junctional conductance in hepatocytes coupled via Cx32 and Cx26 [Saéz et al., 1986, 1990]. Similarly, an increase in junctional conduc-

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tance in response to cAMP has been found in cardiac myocytes coupled via Cx43 [Burt and Spray, 1988; De Mello, 1988]. The changes in conductance are very rapid and occur in several minutes. The action of PKA on rat hepatocyte Cx32 has been attributed to a phosphorylation of Ser-233 which is embedded in a motive (Lys-Arg-Gly-Ser) known to conform with the target sequence for PKA or PKG, i.e. basic-basic-spacer-Ser. The serine can be replaced by threonine [Saéz et al., 1990]. This sequence cannot be found in Cx43 so that it has been hypothesized that Cx43 is not subject to direct phosphorylation by PKA. In accordance with this, Kwak and Jongsma [1996] investigated the influence of 8-Br-cAMP, a direct activator of PKA, on dye coupling and electrical coupling in pairs of neonatal rat cardiac myocytes. They did not observe a change in coupling in response to 8-Br-cAMP. In other tissues as myometrium and Sertoli cells a PKA-dependent decrease in junctional conductance has been found (table 2). Injection of cAMP into canine Purkinje fibers also increased gap junction coupling [De Mello, 1984] within 60–90 s after injection of the nucleotide. The role of cAMP and ATP is further elucidated by the finding that, in double whole-cell patch-clamp experiments, a rundown is normally observed which can be suppressed by addition of cAMP and ATP to the pipette solution, indicating that the spontaneous uncoupling is probably due to washout of intracellular nucleotides [Neyton and Trautmann, 1985; Somogyi and Kolb, 1988a, b]. However, no increase in coupling in neonatal rat cardiomyocytes was seen by Kwak and Jongsma [1996], which is in line with the finding of Berthoud et al. [1993] that 1-hour treatment with 0.5 mmol/l 8-Br-cAMP of MDCK cells, which express Cx43, did not change the immunoblot pattern of Cx43 indicating that 8-Br-cAMP did not induce phosphorylation via PKA of Cx43 in this cell line. In addition, dye transfer through Cx45 gap junction channels and electrical coupling in SKHep1 cells is not influenced by PKA activation [Kwak et al., 1995a]. In the same model, transfectants (SKHp1/ Cx26 and SKHep1/Cx43) were investigated demonstrating that PKA did not influence conductance of Cx43 or Cx26 channels either. However, the possibility that PKA alters the open probability of the channels could not be ruled out in this study since the effects were observed in the presence of uncoupling agents. Additionally, it cannot be fully excluded that in the transfected cell line proteins necessary for the full and normal function of PKA are not expressed. In horizontal cells of turtle and fish retinae, a dopamine-induced increase in intracellular cAMP levels is associated with cellular uncoupling [DeVries and Schwartz, 1989; McMahon et al., 1989] (the connexin isoform involved is not identified). Inhibition of phosphodiesterase with IBMX after stimulation of adenylate cyclase using forskolin resulted in an increase in intracellular

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Table 2. Synopsis of the influence of different protein kinases on cell-to-cell-coupling Protein kinase Connexin Tissue

Permeability

gj

Reference

PKA

0 C

0 C

Kwak and Jongsma [1996] DeMello [1988; 1991] Burt and Spray [1988a] Saéz et al. [1986, 1990] Cole and Garfield [1986] Grassi et al. [1986] Kwak et al. [1995a] Kwak et al. [1995a] Kwak et al. [1995a]

PKC

PKG

Tyr-kinase

43 43

Rat cardiac myocytes Cardiac myocytes

32/26 43 43 45 43 26

Rat hepatocytes Myometrium Sertoli cells SKHep1 cells SKHep1/Cx43 cells SKHep1/Cx26 cells

43 43 43 43 43 ? 45 43 26

Rat cardiac myocytes Cardiac myocytes Rat cardiac myocytes Rat cardiac myocytes Sertoli cells Rat epidermal cells SKHep1 cells SKHep1/Cx43 cells SKHep1/Cx26 cells

43 43 45 43 26

Rat cardiac myocytes Cardiac myocytes SKHep1 cells SKHep1/Cx43 SKHep/Cx26

0 B 0

43

Mouse fibroblasts

B

C B 0 0 0 B

B 0 0 0 C C B

C1 C3,B4 B5

Kwak & Jongsma [1996] Spray and Burt [1990] Mu¨nster and Weingart [1993] Doble et al. [1996] Grassi et al. [1986] Gainer and Murray [1985] Kwak et al. [1995a] Kwak et al. [1995a] Kwak et al. [1995a]

B B 0 B2 0

Kwak and Jongsma [1996] Burt and Spray [1988a] Kwak et al. [1995a] Kwak et al. [1995a] Kwak et al. [1995a]

B C B 0 B B B

Crow et al. [1990]

0>No effect; permeability > as assessed by dye coupling; gj>assessed by measurement of electrical coupling; 1>additional conductance state observed; 2>shift to lower conductance values of the frequency distribution of Cx43 conductances; 3>small conductances favored; 4>frequency of 61 and 89 pS conductance reduced; 5>frequency of 140–150 pS conductance reduced.

cAMP and in a reduction in electrical coupling and dye transfer [Piccolino et al., 1984]. From patch-clamp studies it became evident that single-channel conductance was unaffected, but that the reduced overall conductance can be ascribed to a decreased open probability [McMahon et al., 1989]. In addition to the effect of cAMP or PKA activation alone, different and more complex actions have been observed if such a manipulation is carried out at elevated intracellular calcium concentrations [DeMello, 1991]. In the presence of 6 mmol/l Ca2+, injection of cAMP resulted in a biphasic change

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in gap junction conductance with first an increase followed by a decline in gj which was reversible on application of EGTA [De Mello, 1986a]. Besides this, long-term effects of PKA activation and cAMP are known and are described in the following chapter. It is possible that some of the contradictory findings reported in the literature are due to species or tissue differences, or to differences in the intra- and extracellular calcium concentrations or to different time courses, i.e. if effects after a longer period are observed (let us assume longer than 15 min), besides direct influence on gap junction conductance, an involvement in protein synthesis can also contribute to the total effect. In such cases single-channel measurements can be of great advantage. Another important regulator of gap junction function is protein kinase C (PKC), which is activated via diacylglycerol (DAG). DAG results from inositol lipid hydrolysis by phospholipase C (PLC) and is accompanied by inositol triphosphate release. Activation of PLC-induced inositol hydrolysis is an effect of membrane-receptor activation. Receptors being linked to the PLC system include, among others, the adrenergic a1 receptor, the histamine H1 receptor and the vasopressin V1 receptor. However, it should be kept in mind that noradrenaline, histamine and vasopressin can also activate adenylate cyclase via b, H2 or V2 receptors, respectively. Thus, the effects seen with any of these mediators and probably with many other mediators can be a composite effect. In addition, while inositol triphosphate mediates a transient elevation of intracellular calcium, PKC phosphorylates seryl and threonyl residues at specific sites [Berridge, 1984]. Thus, receptor activation of PLC-linked receptors normally causes a double effect: an increase in intracellular calcium and PKCdependent phosphorylations, both of which can affect gap junction channels. To study the effects of PKC alone, PKC can experimentally be stimulated using phorbol esters such as 12-O-tetradecanoylphorbol 13-acetate (TPA) in comparison to the inactive phorbol ester 4a-phorbol 12,13-didecanoate. However, a number of isoforms of PKC exist in cardiac tissue including PKCa, PKCb, PKCe, PKCn and PKCc (rabbit heart) [Rouet-Benizeb et al., 1996]. Interestingly, only PKCc was found to be located close to the intercalated disks in this study. TPA treatment is assumed to result in a rapid translocation of PKCa and PKCe in cultured neonatal rat cardiac myocytes [Kwak and Jongsma, 1996]. Thus, it might be speculated that not all isoforms contribute to the gap junction regulation and that differences in the subtypes of PKC responsible for the effect may contribute to some of the differences observed between various preparations. Very early in gap junction research an effect of PKC on cellular coupling was observed with TPA on epidermal 3T3 cells. Following TPA administration metabolic coupling between these cells was inhibited [Murray and Fitzgerald,

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1979]. A similar result was obtained using a DAG analogue, 1-oleoyl-2-acetylsn-glycerol, to activate PKC in rat epidermal cells [Gainer and Murray, 1985]. In addition, it was shown that lens cell protein and the liver 27-kD gap junction protein can be phosphorylated by PKC [Dermietzel et al., 1984; Takeda et al., 1987, 1989]. However, there are other reports indicating that Cx26 has no consensus sequence for phosphorylation and is not phosphorylated in isolated liver gap junctions incubated with ATP and the catalytic subunit of cAMPdependent protein kinase, PKC, or Ca2+/calmodulin-dependent protein kinase II [Saéz et al., 1990; Traub et al., 1989; Zhang and Nicholson, 1989], whereas Cx32 [Saéz et al., 1990; Takeda et al., 1987, 1989] Cx43 [Crow et al., 1990; Laird et al., 1991; Musil et al., 1990a] and Cx45 [Laing et al., 1994; Traub et al., 1994] are phosphoproteins. With regard to cardiac myocytes, increases as well as decreases in gj have been observed in pairs of neonatal cardiomyocytes after application of TPA [Mu¨nster and Weingart, 1993; Spray and Burt, 1990]. In addition, Kwak et al. [1995a] found that TPA increases electrical conductance assessed by a doublecell voltage-clamp method, but decreases permeability as assessed by dye coupling in neonatal rat cardiomyocyte gap junction channels. Thus, permeability for small molecules and electrical conductance do not seem to be related to each other under all conditions. In order to investigate this phenomenon in more detail, Kwak et al. [1995a], in a very elegant study, used an expression system to establish the effect of PKC, PKA and protein kinase G (PKG) on single-channel conductance and permeability. The human hepatoma cell line SKHep1, which endogenously expressed low levels of Cx45 and which was not capable of transfering molecules as lucifer yellow from one cell to another under control conditions, was transfected with Cx43 or Cx26. The absence of dye transfer in cells only expressing Cx45 was not influenced by 8-Br-cAMP (PKA activation), TPA (PKC activation) or 8-Br-cGMP (PKG activation). PKC activation by TPA, however, reduced the frequency of 140- to 150-pS conductances in Cx26 transfectant and favored the smaller conductance state of Cx43 channels along with a decrease in the relative frequency of 61- and 89-pS events. This complicated behavior may eventually account for the diversity of results being reported in the literature. In parental nontransfected SKHep1 cells which were coupled via Cx45, activation of PKC induced an additional 16 pS conductance state (together with the 22- and 36-pS conductances observed before). Thus, Cx43 may be regulated posttranscriptionally via PKC-dependent phosphorylation. On the other hand, the influence of PKC on Cx26 channels is expected to depend on another mechanism, yet unknown, since Cx26 is not phosphorylated according to the findings of Traub et al. [1989] and Saéz et al. [1990]. The cGMP-dependent PKG is also involved in the regulation of gap junction channels. Activation of PKG by cGMP or cGMP analogues, such

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as 8-Br-cGMP or b-phenyl-1,N2-ethanoguanosine-3,5-cyclic monophosphate (PET-cGMP), was reported to result in reduced dye coupling and gap junction conductivity in cardiac myocytes coupled via Cx43 [Burt and Spray, 1988a; Kwak and Jongsma, 1996]. In a study on SKHep1 cells and transfected SKHep1 cells Kwak et al. [1995a] found no effect of PKG on Cx26 and Cx45 channels, but a reduction in dye coupling and a shift to lower conductance values of the frequency distribution of Cx43 conductances. As many receptors involved in the regulation of cellular growth are linked to tyrosine kinases, the role of tyrosine kinase in gap junctional coupling and transcellular communication has also been investigated. Since the gene product of the cellular and viral src gene, a 60-kD protein, expresses tyrosine kinase activity, expression of the src gene has been used to investigate the role of tyrosine kinase. Activation of the src gene was shown to reduce dye transfer accompanied by tyrosine phosphorylation within about 30 min [Azarnia and Loewenstein, 1984]. In addition, the stimulation of receptor tyrosine kinases via epidermal growth factor and platelet-derived growth factor in cultures of rat kidney cells and BalbC3T3 cells induced a decrease in dye transfer and in electrical coupling within several minutes [Maldonado et al., 1988]. Crow et al. [1990] described phosphorylation of Cx43 at tyrosine residues in mouse fibroblasts after transfection with Rous sarcoma virus. It has been shown in the developing avian heart as well that tyrosine phosphorylation inhibits Cx43 channels [Veenstra et al., 1992]. Injection of alkaline phosphatase into cell pairs of neonatal rat cardiomyocytes resulted in an enhanced frequency of single-channel events of higher conductance, i.e. of 71.5 pS [Kwak and Jongsma, 1996]. Similarly, Moreno et al. [1994b] observed two conductance states in SKHep1 cells transfected with human Cx43 of 60–70 and 90–100 pS. Depending on the phosphorylation, either the state of smaller or greater conductance was favored. Intracellular injection of alkaline phosphatase preferably led to channel conductances of about 100 pS, whereas inhibition of phosphatases with okadaic acid gave priority to 60 pS events. In accordance with these findings the protein kinase inhibitor, staurosporine (i.e. preventing phosphorylation), induced a higher occurrence of the 100 pS events. These findings indicate that phosphorylation goes along with the increased occurrence of 60- to 70 pS events and dephosphorylation with 100 pS events [Moreno et al., 1992, 1994b]. Several ions are involved in the regulation of gap junctional conductance including Ca2+, Mg2+, H+ and Na+. Very early Ca2+-induced reduction of junctional permeability has been described in Chironomus salivary glands [Rose and Loewenstein, 1975] and heart [De Mello, 1975]. Using the calcium-sensitive fluorescent dye, aequorin, Rose and Loewenstein [1975, 1976] demonstrated parallel changes in pCa and uncoupling, concluding that the actual pCai is

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responsible for uncoupling. However, according to Noma and Tsuboi [1987] intracellular calcium concentrations exceeding about 1 lmol/l, i.e. pCa lower than 6, are needed to affect coupling in guinea-pig hearts. The corresponding pKCa values were 6.6, 6.4 and 5.6 at pH 7.4, 7.0 and 6.5, respectively. At each of these pH values calcium induced uncoupling with a Hill coefficient remaining constant at about 3.4. Lowering the pH shifted the gj-Ca2+ relationship to the right, i.e. higher calcium concentrations were required for half maximal depression of gj . Noma and Tsuboi [1987] concluded from these experiments that Ca2+ and proton compete for negatively charged binding sites at the ‘Ca2+-receptor’ site involved in the control of gj. They hypothesized that the negative charges necessary for calcium binding may be neutralized by the protons and proposed a cooperative receptor model: H2nR ACCB R ACCB CanR, 2n H n Ca

with R>Ca receptor and n>number of Ca-binding sites per receptor, deducing from this the normalized junctional conductance gj to follow the equation: gj>1Ö(1/{1+(KCa /[Ca])n · (1+ ([H]/KH)2n)})

with KCa being the [Ca2+] required for half-maximal depression of gj and KH the [H+] necessary to induce 50% protonation of the receptor. The apparent E half-maximum Ca2+ concentration (KCa ) obtained experimentally is described by the equation: E n n (KCa ) >{KCa · (1+([H] /KH)2n)}.

With the assumption of n>3 Noma and Tsuboi [1987] calculated KCa and KH to be 3.16 · 10Ö7 and 1.12 · 10–7 mol/l, respectively. Since on the other hand the pH-gj relationship was not influenced by Ca2+, Noma and Tsuboi [1987] suggested two types of binding: one for divalent cations, and another for H+. A moderate increase in intracellular calcium concentration obviously does not affect gj in adult heart cells [Ru¨disu¨li and Weingart, 1989, 1991], but higher changes in [Ca2+]i reduce gj in guinea-pig and rat hearts [Maurer and Weingart, 1987]. Maurer and Weingart [1987] concluded from their experiments that reduction in gj occurs if the intracellular calcium concentration exceeds the range of 320–560 nmol/l, which is below the value proposed by Noma and Tsuboi [1987]. Maurer and Weingart [1987] argued that the difference might be due to different stability constants for the calcium buffer used to calculate the cytosolic Ca2+ concentration. It has been suggested that the binding site for Ca2+ and H+ is located on the cytoplasmic loop of Cx43 [Spray and Burt, 1990]. White et al. (1990) showed that rises in [Ca2+]i did not affect gj if pH was maintained at 7.0.

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The 17-kD protein calmodulin acts as an intracellular Ca2+ receptor transducing the Ca2+ signal. A considerable number of the effects are mediated by the activation of Ca2+-calmodulin-dependent protein kinases [Blackshear et al., 1988; Cheung, 1982]. Since the first report on a possible involvement of calmodulin in the regulation of gap junction intercellular communication [Peracchia et al., 1983] calmodulin-binding sites have been identified in a variety of junctional connexins such as Cx43, Cx32 and Cx38 [Girsch and Peracchia, 1992; Peracchia, 1988; Peracchia and Shen, 1993]. With regard to the role of calmodulin it has been shown by Peracchia et al. [1983] that calmodulin inhibitors can prevent cellular uncoupling. Thus, it might be speculated that at least in part the calcium effects may be transduced by Ca2+ calmodulin. As described, the intracellular pH is an important regulator of gj . Intracellular acidification is known to decrease junctional electrical coupling in cardiomyocytes and in Purkinje fibers [Burt, 1987; Reber and Weingart, 1982]. gj was nearly constant in a pH range from 7.4 to 6.5 and decreased sharply when pH was reduced to 5.4 [Noma and Tsuboi, 1987]. This pH-gj relationship was principally not affected by intracellular pCa. They found a Hill coefficient of about 2.4, indicating the number of proton-binding sites per receptor and a half-maximal concentration of 6.1 (pKH). In neonatal rat heart cells Firek and Weingart [1995] found a similar pKH with 5.85. One H+-binding site could be identified as histidine-95 in cardiac Cx43 by Ek et al. [1994]. Hermans et al. [1996] investigated the effects of site-directed mutations in Cx43-transfected SKHep1 cells by exchange of His-126 and His-142 and found an uncoupling effect of acidification related to the position of histidines in the cytoplasmic loop rather than to the total number of histidines. They reported that a fall in pHi caused a reduction in open-channel probability but not in channel conductance. Using the NH3 /NH+ 4 pH-clamp method in Cx43-transfected SKHep1 cells, Cx43 channels close at pH 5.8. The single-channel conductance, however, was not altered (40.8 pS at pH 7.0). In contrast, Cx45 channels in the same expression system closed at pH 6.3. In Cx45 channels the singlechannel conductance (17.8 pS at pH 7.0) did not exhibit pH sensitivity. Thus, the Cx45 channel was concluded to be far more sensitive to changes in pH [Hermans et al., 1995]. Regarding the pH sensor, the carboxy tail length has been demonstrated as a determinant of pH sensitivity [Liu et al., 1993]. Further investigations [Morley et al., 1996] revealed a new model of intramolecular interactions in which the carboxy terminal serves as an independent domain that, under certain conditions, can bind to another separate domain of the connexin protein (e.g. a region including His-95) and close the channel, comparable to the ball-and-chain model for potassium channels. In this receptor (His-95),

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article (COOH-terminal) model, the receptor seems to be conserved among the connexins. An important argument for a more complex mechanism of pH-gating is the delay between intracellular acidification and uncoupling (about 8 min). Delmar et al. (1995) hypothized an action of H+ in association with calcium on the previously phosphorylated connexin. According to Noma and Tsuboi [1987] the KH value on closing the cardiac gap junction is estimated to be in the order of 7.94 · 10–7 mol/l, which is different from that describing the effect of H+ on the gj -pCa relationship (1.12·10–7 mol/l; see above) suggesting different binding sites. In crayfish septate axon, a more complex action of lowering pH has been described [Peracchia, 1991a]: superfusion with Na-acetate led to a rapid increase in junctional resistance (Rj) with a concomitant fall in pHi , but the recovery curve for pHi was slower than that for Rj . A concomitant increase in intracellular [Ca2+] was observed so that it was concluded that the pHi effect on cellular uncoupling in crayfish septate axon is mediated by calcium. Thus, generalizations of the various mechanisms should be avoided and the specific experimental model has to be taken into account. From today’s point of view these acidification experiments may be contaminated by the effects of the pHi -regulating pumps, for example the Na+/H+ exchanger and the Na+/HCO–3 symport as well as other systems, e.g. the Na+/ Ca2+ exchanger [Doering et al., 1996] and possibly the Na+/Ca2+-ATPase. Thus, Yang et al. [1996] described that Na+/HCO–3 cotransport and Na+/H+ exchange contribute to the rate of cell-to-cell electrical uncoupling in ischemic myocardium potentially related to an attenuated Na-dependent calcium loading. In addition, inhibition of proton extrusion with 1 mmol/l amiloride was reported to enhance the effects of changes in pH on gj [Firek and Weingart, 1995]. As a decrease in pHi an elevation in pCO2 can cause a dramatic decrease in coupling in amphibian embryos [Turin and Warner, 1978] and other tissues [Spray et al., 1985]. This has been used experimentally to uncouple preparations. The CO2-induced effect is reversible. The decrease in coupling after exposure to CO2 has been ascribed to the consecutive fall in pHi [Kolb and Somogyi, 1991]. An effect of increasing CO2 in the ventilation air of anesthetized dogs on the cardiac activation pattern has been described and was attributed to gap junctional uncoupling [Vorperian et al., 1994]. However, this cannot be transferred easily on other pathophysiological conditions, since a single increase in pCO2 is seldom, and is often (in pathophysiological situations for e.g. myocardial infarction) accompanied by other changes including depolarization, potassium efflux and many others, which will also affect the sodium channel availability, which will reduce longitudinal propagation velocity. That means that, under pathophysiological conditions, affec-

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tion of the gap junctional channel will probably not be mono- but multicausal. Increasing Mg2+ has also been reported to cause a fall in junctional conductance in pairs of adult guinea-pig cardiomyocytes [Noma and Tsuboi, 1987]. This effect was established for a pMg range from 2 to 3 corresponding to 1 to 10 mmol/l at pH 7.4 in the absence of calcium. The obtained data could be described by the equation: E gj>1Ö(1/{1+(KMg /[Mg])n}),

E E with KMg >3.16 · 10–3 mol/l (pKMg >2.5) and n>3. A Hill coefficient of 3.0 was calculated. Since the slope of the pCa-gj and the pMg-gj relationships were similar, it was suggested that both divalent cations bind to the same receptor site. The uncoupling effect of magnesium has been observed in other systems as well as, for example insect salivary gland [Oliveira-Castro and Loewenstein, 1971]. The cardiac cytosolic free Mg2+ has been measured in the range of 0.48 mmol/l [Murphy et al., 1989] which is below the concentrations in which magnesium induces gap junctional uncoupling. Another ion known to be involved in the regulation of gap junction conductance is Na+. Na+ withdrawal in adult rat cardiomyocytes induced electrical uncoupling as indicated by a decrease in gj /gj · max occurring within 3 min after exposure to 0 mmol/l Na+ [Maurer and Weingart, 1987]. This has been ascribed to the fact that lowering [Na+]0 was reported to result in both an increase in intracellular calcium and a decline in intracellular sodium concentration, which have been interpreted as an impairment in the Na+/Ca2+exchange mechanism, since calcium extrusion via this mechanism requires the transport of sodium [Weingart and Maurer, 1987]. Besides this, De Mello [1976] described an increase in intracellular sodium concentration to cause uncoupling within 500 ms in Purkinje fibers as indicated by an increase in input resistance. It is uncertain whether this was a direct effect of sodium or may be secondary to a rise in intracellular calcium via the Na+/Ca2+-exchange mechanism. As described in the paragraphs above, conclusions on a direct effect of any of these ions should be drawn with caution, since a change in the concentration of any of these may activate a regulatory mechanism to compensate for the change, for example the Na+/H+ exchanger, the Na+/HCO–3 symport or the Na+/Ca2+-exchange mechanism. In addition to ions, other small molecules have been described to play an important physiological and pathophysiological role in the regulation of gap junctional resistance. Thus, ATP acts as an important regulator. In 1979 Wojtcak described that hypoxia in glucose free solution resulted in a rise in Rj in cow ventricular trabeculae indicating that the intracellular ATP content

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may participate in the regulation of gap junctional conduction. Lowering the intracellular ATP concentration down to 0.5 mmol/l from the approximately physiological concentration of 5.0 mmol/l led to a rapid decline in gj [Sugiura et al., 1990] in pairs of adult guinea-pig cardiomyocytes. The investigators kept intracellular calcium and magnesium concentrations at levels less than 10–9 and 0.3 · 10–3 mol/l, respectively. The decrease was reversible upon addition of ATP. Similar to the method used by Noma and Tsuboi [1987], they determined the Hill coefficient for ATP with 2.6 and the half maximum cytosolic ATP concentration in the order of 0.68 mmol/l, suggesting a total uncoupling (gj>0) if ATP is reduced below 0.1 mmol/l. The decrease in gj induced by lowering [ATP] to 0.5 mmol/l was not reversible by adding ADP (10 mmol/l) or 50 lmol/l cAMP or 1 lmol/l of the catalytic subunit of cAMP-dependent protein kinase in these experiments. Thus, at Ca2+, Mg2+ and H+ concentrations considered to be approximately in the physiological range, [ATP] acts as a regulator of gj independent of cAMP-dependent phosphorylation. The authors suggested a direct effect of ATP via a specific ligand-receptor interaction with the gap junctional proteins. Using metabolic inhibition by 2,4dinitrophenol (or decrease to 0.1 mmol/l ATP) in adult guinea-pig cardiomyocytes Morley et al. [1992] also described an increase in gap junction resistance 6 min after addition of 2,4-dinitrophenol. However, the increase in Rj was too small to impair cell-to-cell propagation in this experimental system. With regard to other possible regulators of gj , arachidonic acid, which can be released from membrane phospholipids by activation of phospholipase A2 secondary to activation of a variety of receptors, has been investigated. It became obvious from these experiments that in very high concentrations (50–100 lmol/l) arachidonic acid can evoke cellular uncoupling within several minutes in rat lacrimal gland cells [Giaume et al., 1989]. Since inhibitors of arachidonic acid metabolism did not prevent the arachidonic acid effect, it was suggested that, at least under certain conditions, arachidonic acid (as a fatty acid) may interact directly with the gap junction proteins or their lipid environment. It could be imagined that it is incorporated in the lipid bilayer and alters the geometry of the lipid surrounding of the channels as was suggested for the effect of other lipophilic agents, e.g. heptanol, octanol and halothane. In neonatal rat heart cells, arachidonic acid has also been observed to induce uncoupling [Schmilinsky-Fluri et al., 1990]. This was further investigated by Massey et al. [1992], who described a concentration-dependent effect of arachidonic acid on gj in neonatal rat heart cells in a physiologically more relevant concentration range from 2 to 20 lmol/l. This uncoupling effect could be antagonized by inhibition of lipoxygenase with U70344A, but not with indomethacine suggesting that the arachidonic acid effect at that concentration is mediated by a lipoxygenase metabolite, e.g. a leukotriene, but not by a

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cyclooxygenase metabolite. The incorporation of arachidonic acid in that study was 0.695 mol min–1. Thus, at least two effects of arachidonic acid have to be considered: (1) an unspecific effect in high concentration probably due to physicochemical interaction within the gap junction surrounding, and (2) lipoxygenase metabolites inhibiting the channels in a yet unknown mechanism. Acetylcholine is involved in many aspects of the regulation of the cardiovascular system. Thus, it may also play a role in the control of intercellular communication. Very early in gap junction research the effect of acetylcholine as an important transmitter on gap junction conductance has been investigated. First, Petersen and Ueda [1976] demonstrated an increase in junctional resistance in pancreatic acinar cells following the application of acetylcholine. Concomitantly, the release of amylase was stimulated. A minimum concentration of 1 lmol/l acetycholine was required to evoke uncoupling. The next question was, how is the acetylcholine effect mediated? Calcium has been considered to contribute to the mechanism of action [Iwatsuki and Pertersen, 1978], but does not seem to be the sole mediator as Neyton and Trautmann [1986] demonstrated an uncoupling effect in a double whole-cell technique although calcium was strongly buffered using 20 mmol/l EGTA in the pipette solution. PKC stimulation has been discussed to participate in the transduction of the acetylcholine effect [for a review see, Kolb and Somogyi, 1991]. In rat submandibular gland cells Kanno et al. [1993] demonstrated a reduction in dye coupling from 97.2% (percentage of dye-coupled cells) to 75% and finally 22.7% after application of 10–6 and 10–4 mol/l acetylcholine, respectively. The effect occurred within 10 min. A similar result was found in rat pancreatic acinar cells following the administration of 5 lmol/l acetylcholine, which resulted in cellular uncoupling (dye coupling method) and a 4- to 5-fold increase in amylase release [Chanson and Meda, 1993]. This effect was independent of cycloxygenase, calcium and PKC, but could be inhibited by 1 lmol/l ocadaic acid, an inhibitor of serine-threonine phosphatases, indicating the involvement of a phosphatase in the acetylcholine action. In neonatal heart cells Takens-Kwak and Jongsma [1992] investigated the effect of acetylcholine. They reported a reduction in the intercellular current (Ij) in response to 100 lmol/l of the parasympathomimetic drug, carbachol, which could be mimicked by 8-Br-cGMP. The effect only occurred in the whole cell patch configuration, but not in the perforated patch configuration, suggesting that a cytosolic enzyme is necessary for the effect which is washed out by the pipette in the whole cell patch. Since they found the carbachol effect to be antagonized by alkaline phosphatase Takens-Kwak and Jongsma [1992] concluded that a cytosolic phosphatase is involved in the action of carbachol and, thus, probably of acetylcholine, too.

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Fig. 13. Synopsis of the physiological regulators of gap junction channels. §>PKC changes the substate of conductance, *>not found by all investigators as differences between the various isoforms of connexins or species variabilities.

Another group of transmitters involved in the control of the cardiovascular system by the autonomous nervous system includes the catecholamines, adrenaline and noradrenaline. In acinar submandibular gland cells of the rat the administration of 10–4 mol/l adrenaline elicits a reduction in dye coupling from 97 to 75.3% dye-coupled cells [Kanno et al., 1993]. This could not be mimicked with isoprenaline, but was inhibited with phenoxybenzamine. Thus, the uncoupling effect of adrenaline in this preparation is mediated by stimulation of the a-adrenoceptor, whereas a stimulation of the b-adrenoceptor has no effect. In contrast, stimulation of the b-adrenoceptor in the heart increases intercellular coupling [Veenstra, 1991b]. However, one has to be cautious with generalizations because, following adrenergic stimulation in the intact heart, intracellular calcium and heart rate will also be enhanced, so that a complex effect will occur which is difficult to assess experimentally. Perhaps epicardial mapping experiments measuring the anisotropic ratio, i.e. the ratio between longitudinal and transverse conduction velocity with regard to the fiber axis, will give insight into the global effect of such a manipulation. De Mello [1986b] reported that adrenaline increased the spread of electrotonic potentials during

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diastolic depolarization in canine Purkinje fibers probably due to a rise in intracellular [cAMP]. Very recently an effect of the fibroblast growth factor-2 (FGF-2; a major member of the heparin-binding family of growth factors) on Cx43 in cardiac myocytes has been described [Doble et al., 1996] possibly involving PKC or MAP kinase and tyrosine phosphorylation. The authors showed that incubation with 10 ng/ml FGF-2 for 30 min induces Cx43 phosphorylation on serine residues, with a concomitant loss in intercellular dye coupling and masking of Cx43 epitopes located in residues 261–270. In a previous study it was already shown that basic FGF exists in close association with cardiac gap junctions, and it has been suggested that it, thus, may play a role in gap junctional intercellular communication [Kardami et al., 1991]. FGF-2 can be released, for example, from cardiomyocytes during contraction and after stimulation with catecholamines. The factor is upregulated in response to myocardial damage. In contrast to these findings, FGF-2 induced an increase in Cx43 accumulation and, as a result, an enhancement of coupling between cardiac fibroblasts and capillary endothelial cells, respectively [Doble and Kardami, 1995; Pepper and Meda, 1992]. The mechanisms described so far are synoptically summarized in figure 13. An important point to mention is that, as already said, the affection of the gap junction conductance is not mono- but multicausal under the most physiological and pathophysiological conditions due to the interactions between the intracellular mediators. Thus, most processes will affect intracellular calcium and, on the other hand, a change in intracellular calcium will activate a variety of intracellular mechanisms and affect the activity of many calcium-dependent enzymes.

4.2 Functions in Heart and Vasculature What are the functions of intercellular communication channels in the intact organ? In spite of many details regarding single-channel conductance and the overall conductivity or permeability of gap junctions, their role in the mature and developing heart is currently under intensive investigation and we are probably only at the beginning of an understanding of the role of intercellular communication. Experimentally, it is not possible to measure the gap junction current in an intact heart. However, modern experimental setups will make it possible to get a deeper understanding for the role of these channels in intact tissue. Such setups include mapping experiments using voltage-sensitive dyes like di8-ANEPPS and epicardial potential mapping. With these techniques it is possible to visualize the spread of activation and to measure its velocity.

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An essential role for the normal cardiac development has recently been shown [Reaume et al., 1995] in mouse lacking Cx43 (see chapter 6). Intercellular coupling obviously is a prerequisite for the correct development. Furthermore, synchronization of contraction is facilitated by gap junctional communication as well as synchronization of electrical activation. The electrical coupling between cardiomyocytes mitigates differences in the membrane potential between these cells, for example in the course of an action potential if both cells repolarize at different timepoints. This results in smaller differences in the repolarization times thereby causing a reduction in the dispersion of the action potential duration. Since increased dispersion is known to make the heart more prone to reentrant arrhythmia, sufficient gap junctional communication can be considered as an endogenous arrhythmia-preventing mechanism. For a detailed discussion of the role of gap junctional communication in the biophysics of cardiac activation as related to anisotropy, nonuniformity and stochastic phenomena, see chapter 1; for a discussion of their role in arrhythmia, see chapter 6, and for a possible pharmacological intervention at the gap junctions for suppression of arrhythmia, refer to chapter 7. As already pointed out (see chapter 3), in some specialized areas of the heart, e.g. sinus node, there are special interdigitating patterns of gap junctions, providing some form of isolation from the hyperpolarizing influence of other cells, for example the surrounding atrium. Interestingly, these gap junctions are made from Cx40, which is more sensitive to the transjunctional voltage than Cx43 channels, thereby providing better isolation if higher differences in the membrane potential occur. However, such a physiological function can be imagined but has not been shown directly, yet. The communication between cells via gap junctions can also provide exchange of small molecules and has been shown to protect cells against oxidative stress by exchange of glutathion [Nakamura et al., 1995]. Exchange of small molecules may have more functions when considering signalling molecules such as cAMP or Ca2+. However, there are no experiments available at present on the role of gap junctions in intact organs. Apart from providing communication, gap junctions can, on the contrary, have the function to isolate cells from their surrounding. Such isolation by closure of gap junctions in certain pathophysiologic conditions occurs, for example, in the course of hypoxia [Wojtcak, 1979] and loss of ATP or during myocardial ischemia. This may have the advantage that these cells no longer communicate and participate in myocardial contraction, thereby saving energy, although this has not yet been demonstrated. In the vasculature release of local mediators such as endothelin, prostacyclin and nitric oxide can only affect the local vasotone (at the site of release) or regulate downstream constriction or dilation. Besides this, upstream regula-

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tion has been observed, but not well understood. Often this was ascribed to the influence of the perivascular nerves. However, since Beńy and Connat [1992] showed that smooth muscle cells of the media of pig coronary arteries are dye coupled via single hydrophilic channels, upstream regulation of vasoactivity and transmural regulation (from the luminal side to the periphery and in the opposite direction from the perivascular nerves to the lumen) has been also considered to be influenced or provided by gap junctional coupling (fig. 12).

4.3 Electrophysiology and Voltage-Dependent Gating In general, with regard to the current-voltage relationship there are two types of gap junction channels to distinguish: (a) channels with rectifying behavior, and (b) nonrectifying channels. In addition, the gap junction channels can be distinguished by their single-channel conductance. The single-channel conductance of a given gap junction channel made of one connexin isoform, however, may exhibit different substates of single-channel conductance. In general, the following states can be distinguished: open states with (a) main open state, (b) several substates, (c) residual state, and (d) the closed state. Furthermore, it is important to differentiate between channels exhibiting sensitivity to transjunctional voltage and channels being more or less insensitive. For investigation of the current-voltage relationship there are principally three possibilities: (a) investigation of freshly dissociated cells with the advantage that these cells are isolated from an intact tissue and should resemble the properties of these postmitotic cells rather well, and with the disadvantage that in the course of some hours many of the channels are internalized or dissociate so that the investigator observes a so-called ‘run-down’ with regard to the coupling in such cell pairs; (b) investigation of cultured cells, e.g. embryonic chick heart cells or neonatal rat cardiomyocytes or others, and (c) investigation of gap junctional channels in transfected cells, e.g. SkHep1 cells, tumor cell lines or xenopus oocytes. In such elegant transfection systems a problem may occur if regulatory processes are to be investigated which involve pathways not present in the transfected cell. To investigate the current-voltage relationship in any of the given systems, the standard method is the double-cell voltage-clamp technique [Spray et al., 1981, 1985; Weingart, 1986] carried out as a double whole-cell patch as described by Giaume [1991]. The principle is that a transjunctional voltage difference (by clamping the two cells to different potentials) is applied for a short time to a pair of coupled cells and the current necessary for maintenance of the voltage difference is measured. In order to achieve such an experimental

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Fig. 14. Experimental setup for double whole-cell patch measurement of the gap junction current.

setup two voltage-clamp amplifiers are connected via a patch-clamp pipette to either cell (fig. 14). While in one cell the membrane potential is kept for example at –40 mV (in order to inactivate the sodium current), the membrane potential of the other cell is set for example to –30 mV, thereby applying a transcellular voltage of 10 mV. In this way transcellular voltages ranging from –50 to +50 mV or from –100 to +100 mV are applied and symmetry is controlled by alteration of the cell being kept at –40 mV. Since current can flow across the cell membrane and across the junction between both cells, the current in cell 1 I1 can be described under these conditions by the equation: I1>(V1 /rm1)+[(V1ÖV2)/rj]

and accordingly the current in cell 2 by I2>(V2 /rm2)+[(V2ÖV1)/rj],

with V1 and V2 being the voltage relative to the holding potential VH (e.g. –40 mV in order to inactivate the sodium current) and rm1 and rm2 the membrane resistance in cell 1 and 2, respectively. If cell 2 is kept at VH , currents I1 and I2 can be described as: I1>(V1 /rm1)+(V1 /rj) I2>V1 /rj

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so that I2 is a direct measure of the current flowing across the junctional membrane. Problems may arise from the series resistance of the pipettes and, in some preparations, eventually from the cytosolic resistance or from the ratio between these different resistances (for more details see chapter 8). The current measured is then plotted against the transcellular voltage Vj . Linear regression reveals the total intercellular resistance. gj follows the equation: gj>Ncj P0 ,

with N>number of channels capable of opening and closing, P0 being the open probability and cj the single-channel conductance. If, under the experimental conditions described above, cells are progressively uncoupled by application of, e.g., heptanol or halothane, it is possible with some types of amplifiers to observe single-channel openings shortly before total uncoupling occurs. Alternatively gap junction channels can be reconstituted in lipid bilayers for observation of single-channel conductances. More details and protocols are given in chapter 8. Looking at the current measured using such a protocol with a pulse duration of about 2 s, one can distinguish two components of the junctional current: (a) the instantaneous component, and (b) the steady-state component. Plotting the instantaneous current Ij versus Vj may reveal a linear relation as shown by Veenstra et al. [1993], which means that the instantaneous gap junctional conductance is constant, i.e. gj>Ij /Vj>constant. A linear current voltage relation under such conditions means that the junctional channel behaves like an ohmic resistor with a constant resistance which is insensitive to the transjunctional voltage. However, not in all cases an ohmic behavior will be seen. If with increasing transcellular voltage the current does not follow a linear relation, some kind of rectification is present. Rectification means that the channel resistance increases or decreases with increasing or decreasing transjunctional voltage, i.e. the channel favors a current in one direction or at a special range of transcellular voltage. In other words it behaves comparable to some kinds of diodes. Investigation of the steady-state current and the steady-state conductance is normally carried out by fitting the normalized gss /Vj relationships with the two state Boltzmann distribution which follows the function: gss /ginst>{(gmaxÖgmin)/(1+exp[A(VjÖV0)])}+gmin

according to Spray et al. [1981] and Veenstra et al. [1993] with gmax being the maximum conductance (>1, normalized to instantaneous gj) and gmin the minimum conductance. V0 is the half-inactivation voltage where gj is between gmin and gmax and A>zq/kT (z>number of equivalent electron charges, q> voltage sensor, k>Boltzmann constant, T>temperature).

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a

b Fig. 15. Typical example of a measurement of junctional conductance. For details see text. (Freshly isolated adult-guinea pig cardiomyocytes, holding potential –40 mV, series resistance was overcome by using switch-clamp amplifiers (SEC05).) For pipette solution, etc., see chapter 8.

Using the technique of double whole-cell patch reveals data as shown in figure 15. The holding potential of both cells is –40 mV. One cell is then clamped to –50 mV while the other cell is kept at the holding potential thereby applying a transcellular voltage of 10 mV. The other tracks show the currents necessary to maintain these voltages. Transcellular voltages

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ranging from –50 to +50 mV were applied. From these experiments the I/V relationship in figure 15b could be constructed. A critical problem with such measurements is the compensation of the series resistance since in the case of the junctional resistance being in the order of the series resistance of one of the patch pipettes, distortion of the measurement of the intercellular current will result. When considering the question whether gap junctional channels are regulated by transcellular voltage or not, the reader may be confused by the various findings of different groups in various preparations. However, as a general rule considering cardiac gap junction channels coupled by Cx43 channels, in cell pairs of adult cardiac cells, which are coupled by large numbers of gap junction channels, the instantaneous and the steady-state current-voltage relations have been demonstrated to be linear, i.e. gj is independent of transjunctional voltage and rectification is not observed [Noma and Tsuboi, 1987; Reverdin and Weingart, 1988; Weingart, 1986; White et al., 1985]. In contrast, in embryonic cells (or with some limitations in neonatal cells) which communicate by only a few gap junction channels, linear current-voltage relationships for instantaneous and steady-state gj are observed in a discrete transcellular voltage range: ×50 mV (neonatal rat cardiomyocytes) [Rook et al., 1988]; ×30 mV (embryonic chick heart cells) [Veenstra, 1990], and ×60 mV (neonatal hamster cardiomyocytes) [Veenstra, 1990]. Outside this range rectification is observed and the slope of gss declines progressively to values near zero [Rook et al., 1988; Veenstra, 1990, 1991a], whereas the instantaneous gj remains constant. This voltage-dependent behavior of gss can be fitted with the two-state Boltzmann equation described above. What is the basis of this voltage-sensitive gating? According to Rook et al. [1988] it is not the result of a change in the single-channel conductance cj but in the ratio topen /tclosed , which is decreased so that at a given time point more channels are in the closed state. Spray et al. [1985] found gj to be unaffected by the transjunctional potential gradient and by the membrane potential in dispersed and reaggregated rat ventricle cells. Similarly, Kameyama [1983] reported that Rj is independent of the transjunctional potential gradient. While the instantaneous gj is insensitive to the transjunctional voltage, in most cases gss can exhibit voltage dependence in some preparations [Veenstra et al., 1993] (fig. 16). Voltage dependence in neonatal cardiac myocytes can especially be observed with large transjunctional voltages in the order of 80 mV or more. Rectifying behavior was observed in crayfish axons with depolarization at the presynaptic side increasing the junctional conductance [Furshpan and Potter, 1959; Giaume et al., 1987] and in fish [Auerbach and Bennett, 1969]. It has been hypothesised by Bennett et al. [1991] that this rectifying behavior may arise from a heterotypic composition of the channel.

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a

b

Fig. 16. Voltage-dependent gating in pairs of rat Cx43 transfected RIN cells (a) and pairs of mouse Cx40 transfected HeLa cells (b) [Banach and Weingart, 1996; Bukauskas et al., 1995].

Do all connexins exhibit the same sensitivity to the transjunctional voltage? Trying to answer this question, Nicholson et al. [1993] have shown in an xenopus oocyte expression system that gss of gap junction channels constituted of Cx37 are more voltage-sensitive than those made from Cx40. In comparison to these, Cx32 was more insensitive and Cx26 channels exhibited the minimum sensitivity. In addition, Veenstra et al. [1993] showed that the voltage sensitivity of gss of embryonic chick heart cells decreased with the age of embryos. In Cx40-transfected neuroblastoma cells (N2A) the Boltzmann half-inactivation voltage was determined with –54 and +47 mV at negative or positive Vj, respectively, indicating a sensitivity of gss of the Cx40 channel to transjunctional

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Table 3. Boltzmann equation parameters for various connexins Connexin

Gmin

V0 [mV]

Slope factor

References

37

0.27

×28

0.08

Reed et al. [1993]

40

0.33/0.28 0.32

–54/+47 ×35

0.13/0.11 0.225

Beblo et al. [1995] Ebihara [1995]

43

0.37a

×60

0.106

Moreno et al. [1995]

45

0.072 0.17/0.16

×13.4 –16/+22

0.115 0.126/0.115

Moreno et al. [1995] Ebihara [1995]

If two values are given, the data on the left are mean values at negative Vj and on the right mean values at positive Vj . It becomes evident that Cx43 exhibits the lowest sensitivity to Vj since the half inactivation voltage V0 is highest, whereas according to these data Cx45 and Cx37 exhibit the strongest Vj dependence. a Gmin /Gmax .

voltage [Beblo et al., 1995]. The Cx43 channel has been shown to possess a voltage-sensitive component as well with Boltzmann half-inactivation voltages at –69 and +61 mV [Wang et al., 1992] (table 3). In a recent study on mouse Cx40 transfected HeLa cells, Bukauskas et al. [1995] determined V0 –45/49 mV and gmin 0.24/0.26. Valiunas et al. [1997] investigated the dependence on transjunctional voltage in neonatal rat cardiomyocytes, which are normally coupled via Cx43, and found, depending on the pipette solution used, V0 –51/51 mV, gmin 0.28/0.25 and z 3.1/2.9 (KCl solution), or V0 –59/59 mV, gmin 0.15/0.16 and z 2.0/2.3 (TEA-aspartate solution). In another detailed study Banach and Weingart [1996] observed asymmetrical-gating properties in rat Cx43-transfected RIN cells with V0 –73.7/65.1 mV and gmin 0.34/0.29, if an asymmetric protocol was used (i.e. one cell is kept at the holding potential and the other is stepped to different voltages thereby applying a transjunctional voltage), whereas if a symmetrical protocol was used (both cells are clamped to the holding potential and then a certain voltage clamp step is applied but of opposite polarity in both cells), the authors observed symmetrical gating with V0 –60.5/59.5 mV, gmin 0.27/0.29. The problems arising from the various findings regarding the voltage sensitivity of gss initiated a very elegant study by Jongsma et al. [1993] answering the question ‘Are cardiac gap junction channels voltage sensitive?’. In a computer simulation they modelled two cardiomyocytes interconnected via a gap junction and varied the number of gap junction channels within this intercon-

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nection. The open probability of a single channel was assumed to follow the equation: p0>1/{1+exp([Aa+Ab][dVjÖV0])},

with a>k exp(ÖAa[dVjÖV0]) and b>k exp(ÖAb[dVjÖV0]) and Aa (>0.041 mV–1) and Ab (>0.021 mV–1) being the voltage sensitivities of a and b, k (>2.5 s–1), the rate at which a equals b and V0 (>74 mV), the transjunctional voltage at which a equals b (the data were obtained from single-channel measurements carried out on neonatal rat heart cells by Rook et al. [1988]). They found a relationship between the pipette series resistance and the appearance of voltage insensitivity of gss . It was shown that gap junctions with intermediate numbers of channels (130 or more channels were assumed) appear to be voltage insensitive if pipettes with 60 MX are used and gap junctions with 300 or more channels if 20 MX pipettes are used. In real measurements the circuit is even more complicated by the fact that, part of the junctional current is shunted to ground through the membrane resistance. The higher the series resistance the more difficult it is to detect sensitivity. In addition, they demonstrated that, in cells well coupled by large numbers of gap junction channels (as in real experiments often in adult cells), voltage sensitivity cannot be detected or is only very moderately present, whereas in cells weakly coupled by only a few channels sensitivity to transjunctional voltage can be observed. Thus, Jongsma et al. [1993] concluded that the ‘cardiac gap junctions are moderately voltage sensitive’ and that the decrease in voltage sensitivity in the course of embryonic development as described by Veenstra [1991b] does not reflect a change in the regulation of junctional resistance but rather an increase in mean gap junction size. Finally, they state that, in real experiments on well-coupled heart cell pairs, this voltage sensitivity of gss cannot be observed mainly because of the presence of gap junction channel access resistance and pipette series resistance. Much has been speculated about the possible role of a sensitivity for transjunctional voltage in the heart. It can be assumed that such a behavior would protect a cell from the hyperpolarizing or depolarizing influence of other cells provided the transjunctional voltage is high enough, i.e. exceeding for example 50 mV in rat heart cells (see above). This would be necessary for sinusnodal cells which are in close vicinity to the atrium which is relatively hyperpolarized with regard to the sinus node. Similarly, AV node cells adjacent to non-nodal tissue may be subject to such an influence. In these cells potential gradients high enough to disturb for example the pacemaker function may arise and, thus, such a disturbing influence may be prevented by transjunctional voltage-sensitive gating of the gap junctions. This fits with the general finding that Cx40 and Cx45 are more sensitive to transjunctional voltage than Cx43. However, there is at present no clear evidence for these hypotheses.

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What is the real range of gap junction resistance measured in various systems? Kameyama [1983] reported in reaggregated cell pairs of adult guineapig hearts Rj in the order of 1.4–2.1 MX (corresponding to 476–714 nS) and Noma and Tsuboi [1987] 0.25–11 MX (90–3.900 nS) in adult guinea-pig cardiomyocytes with a peak in the distribution around 1,000 nS (i.e. 1 MX). Weingart [1986] calculated the gap junction conductance in adult cardiomyocytes with 204 nS (4.9 MX). In neonatal or embryonic heart cell pairs lower conductances were reported: 0–30 nS (?33 MX) in embryonic chick heart cells from 7-dayold embryos [Veenstra, 1990] and 0.05–35 nS (?28 MX) in neonatal rat heart cells [Burt and Spray, 1988a]. In cable preparations of various species resistance has been reported in the order of 200–600 Xcm: 523 Xcm in bullfrog trabeculae [Haas et al., 1983]; 200–250 Xcm in guinea-pig trabecular muscle [Daut, 1982]; 350–530 Xcm in rabbit Purkinje fibers [Colatsky and Tsien, 1979] and 588 Xcm in frog ventricular trabeculae [Chapman and Fry, 1978]. An interesting phenomenon is that some of the voltage-insensitive gap junctional channels are gated, i.e. the channels open and close rather than remain open all the time. The mechanisms underlying this gating behavior of voltage-insensitive channels found in avian and mammalian hearts and in septate axons of earthworms are still unknown [Brink, 1991]. Since as pointed out above the overall gap junction conductance does not only depend on the number and the open probability of the channels but also on the single-channel conductance, single-channel conductance of various connexins, including Cx37, Cx40, Cx43, Cx45, will be discussed. From the channel geometry with a pore sink diameter of 1.5 nm, a pore mouth diameter of 2.3 nm and a pore length of 15 nm (values according to Makowski [1985] and Zampighi [1987]), Ru¨disu¨li and Weingart [1989] predicted the single-channel conductance. According to their considerations and to Hille [1992], RChannel is given by the equation: RChannel>RPore+RAccess

with RPore>q(1p(d/2)2)

and RAccess>2q/pd.

Assuming the inner pore to be filled with a 130-mmol/l salt solution q equals 100 Xcm and RChannel equals 10–20 GX. The single-channel conductance was predicted with 50 to 100 pS. However, it has been suggested and shown by various investigators [Loewenstein et al., 1978; Neyton and Trautmann, 1985;

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Rook et al., 1988] that the regulation of gap junction conductance is not the all or nothing and not quantal, but graduate so that different single-channel conductances or substates have been postulated [Page, 1991]. This is probably reflected by various substates of single-channel conductance found in various gap junctional channels which will be discussed as well. The channel found most abundantly in cardiac tissue is the Cx43 channel. In guinea-pig hearts the single-channel conductance of Cx43 channels has been measured with 37 pS and it was characterized as insensitive to the nonjunctional membrane potential [Ru¨disu¨li and Weingart, 1989]. In contrast in the neonatal rat heart Burt and Spray [1988a] found a single-channel conductance of about 60 pS. The voltage-insensitive component of gj of Cx43 channels has been ascribed to a voltage-insensitive substate by Moreno et al. [1994a] who observed a graded response in Cx43 transfected hepatoma cells. In another study [Moreno et al., 1994b] these authors defined two substates of this channel, i.e. 60–70 pS and a higher conductance state with 90–100 pS. Kwak et al. [1995b] also characterized two substates in neonatal rat cardiomyocytes with 20 and 40–45 pS. However, when taking the regulation by protein kinases into account Takens-Kwak and Jongsma [1992] discriminated even three substates of single-channel conductance in neonatal rat heart cells: 21, 40–45 and 70 pS. Similarly, in Cx43-transfected SKHep1 cells they found three different substates: 30.5×9.1, 61.2×9.8 and 89.1×12 pS [Kwak et al., 1995a]. From these different findings one might conclude that at least three different substates of single-channel conductance of Cx43 channels can be distinguished. Depending on phosphorylation or dephosphorylation by various protein kinases the different substates seem to be favored (see chapter 4.1). As investigated by Valiunas et al. [1997] gap junction channels of neonatal rat heart cells formed by Cx43 possess several conductance states: a main state, several substates, and a residual state as well as a closed state. Depending on the pipette solution cj (main state) was determined 96 pS (KCl), 61 pS (Cs-aspartate) and 19 pS (TEA-aspartate) and cj (residual state) was determined 23, 12 and 3 pS, respectively, revealing cj (main state)-cj (residual state), ratios of 4.2, 5.1 and 6.3, indicating that the residual state restricts ion movement more efficiently than the main state. Transitions between the several open states (main, substates and residual state) were fast (=2 ms) in contrast to the transitions between open states and closed state (15–65 ms). The differences in the results of various investigators may perhaps be due to different experimental models and conditions. An example of single-channel recordings is given in figure 17. The next channel to be dealt with is the Cx40 channel. The unique conductance and gating of gap junction channels formed by Cx40 has been investigated in Cx40-transfected mouse neuroblastoma cells (N2A cells). In

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Fig. 17. Single-channel conductance of neonatal rat heart cells. Note the residual conductance [Valiunas et al., 1997]. The Vj applied was 50 mV.

the presence of potassium glutamate (120 mmol/l) Beblo et al. [1995] measured the slope conductance of single Cx40 channels in the order of 158 pS. The macroscopic steady-state current exhibited dependence on transjunctional voltage with a Boltzmann half-inactivation voltage of ×50 mV. The authors found a residual voltage-insensitive normalized junctional conductance in the order of 35% of the maximum and a gating charge valence of 3. In these Cx40 channels substates have also been described [Beblo et al., 1995] equal to 21 and 48% of the main open-state conductance (which was in the range of 158 pS), although these substates were reported to occur only occasionally. Bukauskas et al. [1995] investigated mouse Cx40-transfected HeLa cells and measured single-channel conductance. They observed three open states: main state (198 pS); several substates, and an residual state (36 pS) besides a closed state. Transition between the open states were fast (1–2 ms) in contrast to the transitions between open and closed states (15–45 ms). The third channel found in heart muscle is the Cx45 channel. This was reported to exhibit an overall conductance of 3.1×0.4 nS in cell pairs [Kwak et al., 1995a] with a mean single-channel conductance of 36.5×6.5 pS or 1.3 nS in human Cx45-transfected SKHep1 cells [Moreno et al., 1995] and cj of 32×8 pS. At higher transjunctional voltages an additional conductance state with 22.5×4.3 pS was observed. This channel strongly depends on transjunctional voltage [Moreno et al., 1995] comparable to Cx38 channels. However, single-channel conductance is not a function of transcellular voltage. Besides Cx40, Cx43 and Cx45, Cx37 channels are expressed in the cardiovascular tissue. These channels are frequently found in endothelium. These channels were expressed in human Cx37-transfected neuroblastoma cells (N2A cells) and exhibited a pronounced voltage dependence and multiple conductance states [Reed et al., 1993]. Several single-channel conductances were found:

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219×22, 165×6, 123×13 and 53×1 pS at Vj>30–40 mV. At higher Vj the large conductance was no longer observed and cj of 94×4, 69×5 and 39×10 pS were detected at Vj>80–90 mV. Finally, Cx26 channels were also investigated in an expression system (SKHep1 cells) by Kwak et al. [1995a]. They observed a mean single-channel conductance of 140 to 150 pS with substates of 70 and 110 pS. Gating of the channel can be modulated either by an alteration in the single-channel conductance or by a change in the open probability. The macroscopic channel conductance is then either influenced by the single-channel conductance, by the mean open time of a channel or by the frequency of channel openings, this means by the number of channels in the open state at the same time or at a given time interval. Do the gap junctional channels exhibit some sort of selectivity? Do they conduct cations and anions? Do they exhibit characteristics known from so many ion-selective transmembrane channels or are they distinct from these? As already said, the first important difference to other ionic channels is that gap junction channels are permeable to small molecules up to a molecular weight of about 1,000 Daltons. Another difference is that they conduct both anions and cations. In order to further elucidate these questions Brink [1991] investigated the selectivity of gap junctional channels in septate earthworm axons using the double whole-cell patch-clamp technique. According to this study this channel exhibits a single-channel conductance of about 100 pS and is sensitive to calcium and pH. There was no inhibition of the channel conductance with various blockers commonly used in electrophysiology: tetraethylammonium (TEA), 4-aminopyridine (4-AP), Zn2+, Co2+ and Ni2+. There was no selectivity for K+ over Cs+, but the 100-pS channel seemed to be somewhat selective for cations over anions with a chloride conductivity to potassium conductivity ratio of 0.53. This channel is also voltage-insensitive. Molecules not larger than 1 kD or not exceeding an ionic radius of 0.8 or 1.0 nm can be transported through the channel [Brink, 1991; Spray et al., 1991]. The rat Cx40 channel exhibits a detectable chloride permeability of 0.29 relative to potassium [Beblo et al., 1995]. This indicates some selectivity for cations over anions as well. These channels were also permeable to 2,7dichlorofluorescein and to the more polar 6-carboxyfluorescein dye. Interestingly, the 2,7-dichlorofluorescein permeability did not increase with increasing junctional conductance in that study. With regard to small molecules other than ions Tsien and Weingart [1976] reported that 3H-cAMP diffuses across gap junctions in calf and cow ventricle. Furthermore, Weingart [1974] has shown that 14C-TEA (molecular weight 130) diffuses transjunctionally in sheep ventricular muscle and he found the channel

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˚ . A number of different dyes with molecular diameter to be in the order of 10 A weights ranging up to 859 Daltons have been demonstrated to diffuse across the gap junctions including procion yellow (MW 697) in sheep and calf Purkinje fibers [Imanaga, 1974], 6-carboxyfluorescein (MW 670; diffusion coefficient 5.8 · 10–6 cm2/s), lucifer yellow (MW 457; diffusion coefficient 3.0 · 10–6 cm2/s), lissamine rhodamine B200 (MW: 859; diffusion coefficient 8.6 · 10–7 cm2/s), while Chicago blue (MW 1,000 Daltons) did not diffuse across the channels [Imanaga, 1974, 1987; Imanaga et al., 1987]. From these data the authors calculated an upper limit for the channel diameter of 1.2–1.3 nm. In more physical terms the permeability Pj through a gap junction channel can be described using the equation: Pj>Vcell ki /Aj ,

with Aj being the area of gap junctional membrane, Vcell the cell volume and ki the experimentally measured rate constant for transcellular diffusion. Thus, the gap junction channel behaves like a gated pore exhibiting some selectivity for cations over anions, and acting as a diffusion barrier for molecules exceeding 1,000 Daltons. It does not show the high selectivity for any sort of ion as known from other ionic channels. With regard to voltage sensitivity, it behaves like an ohmic resistor as far as the instantaneous Gj is concerned. The steady-state conductance Gss can exhibit a more or less stronger sensitivity to Vj depending on the connexin the channel consists of.

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5

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Regulation of Gap Junction Expression, Synthesis and Assembly

We have considered the structure, diversity and the function of gap junctional channels. But, how are gap junctional channels formed, how are they degraded? Or are they not subject to any turnover? Little is known at present on the process of channel formation and assembly. And even less on the regulation of channel formation. A very interesting and important question is: how can two cells direct their hemichannels so that they fit each other forming the intercellular pore? Research in this area is still at its beginning and many of the processes involved are not well understood. But nevertheless, in this chapter the present knowledge on the regulation of channel expression and turnover will be summarized. Gap junctions and their channels are not static; as many other cell proteins they underlie a turnover. For example it has been shown in the human neonate and child that there is a progressive polarization of the gap junctions towards the positions of the mature intercalated disks reaching the adult pattern at an age of about 6 years [Peters, 1996]. Thus, the pattern of gap junction expression can change with time as has been shown in various diseases, for example in the course of chronic myocardial infarction and heart failure (see chapter 6). On the other hand there is a considerable change in the expression of gap junctions during development as shown, for example, in the developing avian embryonic heart [Veenstra, 1990]. The basis of such alternating patterns must be a turnover process with assembly and degradation of gap junctional channels. If cells come into contact the occurrence of cell coupling has been detected using dye transfer and electrical methods. This raises an important question: is rapid de novo synthesis required for the formation of gap junctional channels? Answering this question Epstein et al. [1977] inhibited the protein synthesis by treatment with cycloheximide and subsequently brought cells into close contact. They observed progressive cellular coupling indicating that for the formation of gap junction channels no rapid de novo synthesis is necessary. This implies that presynthesized channels must be stored within a cell which then can form the channels. An alternative hypothesis is that hemichannels are incorporated into the membrane under normal conditions and that after coming into contact with another cell the hemichannels of both cells are translocated in order to form channels bridging the gap by ‘interlocking’ of hemichannels.

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It has been demonstrated that cell coupling is established within 3–30 min after bringing disaggregated cells into contact. Interestingly, the resulting increase in cell-to-cell conductance proceeds in quantal steps [Loewenstein, 1981; Loewenstein et al., 1978] which may be interpreted by the progressive assembly of channels increasing the conductance stepwise with the formation of every channel. However, it is not certain whether this is the correct interpretation or whether this is an oversimplification. Loewenstein [1981] has elaborated this hypothesis to the ‘self-trap’ model: precursors of the gap junction channels, either as hemichannels or as polypeptide constituents of these hemichannels (so-called ‘protochannels’), are present in the plasma membrane of both cells and can diffuse freely within the plane of the plasma membrane lipid bilayer. If cells come into contact, such hemichannels or their precursors can also come into close contact by chance. If they are close enough so that their extracellular loops E1 and E2 can form noncovalent bonds (by van der Waals forces) the extracellular domains of the protochannels interlock and form the complete gap junction channel. If neonatal rat heart cells are manipulated into contact, Valiunas et al. [1997] observed new gap junction channel formation at a rate of 1.3 channels/ min (the first opening occurred within 7–25 min after physical cell contact). They argued that this formation occurs by docking of preformed hemichannels of adjacent cells. How are gap junctions synthesized and incorporated into the plasma membrane? Normal plasma membrane proteins are synthesized at the ribosomes of the endoplasmatic reticulum (ER) and cotranslationally inserted into the membrane. This is followed by posttranslational folding and eventual oligomerization. Thereafter, the molecules are transported through the Golgi apparatus and carried to their final position in the plasma membrane. Falk et al. [1995] investigated this process for rat liver, dog pancreatic and baby hamster kidney gap junctions. They could identify nearly the same pathway for gap junction assembly as described above for other membrane proteins. However, the integration of the gap junctions into the ER membrane requires an additional ‘assisting factor’, which is most likely a cytoplasmic chaperonlike protein. Chaperones are cytoplasmic proteins which are involved in protein folding and assembly. Together with the chaperonines they help to give the newly synthesized proteins their final structure [for review see, Hartl, 1996]. The binding of this putative assisting factor was suggested to occur at the NH2 terminus of the gap junction protein anchoring the NH2 terminus to the cytoplasmic site of the ER membrane. Using the metabolic inhibitor monensin, Puranam et al. [1993] found an intermediate form of Cx43 in the Golgi of rat cardiac myocytes. Cx43 entered and accumulated in the Golgi network of monensin-treated cells. Further investigation revealed that one accumulating

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form was the phosphorylated state of the nascent 40-kD form of Cx43 suggesting an early phosphorylation of the Cx43 protein in the secretory pathway. Furthermore, in vivo studies revealed that the further assembly of the hemichannels formed in the Golgi network depended on protein phosphorylation and the presence of adhesion molecules [Musil and Goodenough, 1993]. Using Cx43 in rat kidney cell cultures Musil and Goodenough [1995] found that the connexon formation occurs after transport through the cis, medial and trans Golgi cisternae, since the connexon assembly could be blocked by brefeldin A, a specific blocker for assembly processes occurring before or at these compartments. The authors concluded that the connexon assembly takes place in the trans Golgi network in contrast to other integral membrane proteins. Secondary to the formation of connexons as oligomeres from connexins the association of a connexon in the plasma membrane in one cell with a connexon in an adjacent cell membrane to form the intercellular channel has to be considered. Miner et al. [1995] could demonstrate a regulatory role for the cadherins in the gap junction assembly: calcium-dependent adhesion proteins (cadherins) have been shown to have a significant influence on gap junction assembly, since in disaggregated cells the formation of intact intercellular channels can be inhibited by Fab fragments of N-cadherin-specific antibodies. If cadherins are involved, a calcium dependence of the gap junction assembly process should be detectable. In Novikoff hepatoma cells expressing Cx43, Miner et al. [1995] investigated the sensitivity of the gap junction formation on extracellular calcium by means of electron microscopy and a dyetransfer technique. It became obvious that the percentage of coupled cells after reaggregation was decreased with a reduction in extracellular calcium concentration from 1.8 nmol/l to 40 nmol/l in a nonlinear fashion. Besides this, no change in phosphorylation was observable. Two conclusions from these findings were made by the authors: on the one hand one can suggest a simple approximation of the two plasma membranes as the prerequisite of intercellular channel formation and on the other hand a signalling process between calcium, cadherins and gap junction proteins can be imagined. The latter is supported by the finding of a nonlinear relationship between calcium and gap junction formation. Fishman et al. [1991b] looked at the expression of Cx43 in the developing heart and found accumulation of Cx43 mRNA during embryonic and early neonatal stages accompanied by a temporally delayed increase in the protein. With maturation of the heart these levels decline suggesting that increases in intercellular coupling characterizing cardiac development do not solely depend on modulation of Cx43 gene expression but also involve formation of functional gap junction channels within the intercalated disks. One might speculate from the above findings that such

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processes might be regulated via cell adhesion molecule signalling. However, at present these possible interactions have not been investigated. Thinking about the signalling and the regulation of gap junction assembly one might consider the cytoskeleton to be involved but, at present, gap junctions have not been reported to be attached to the cytoskeleton. During the development of the human heart it has been found that there is a close and increasing association between the gap junctions and the fascia adherens junctions [Peters et al., 1994]. While in the neonate Cx43 exhibits a punctuate distribution over the entire surface of the cardiomyocytes, during postnatal development Cx43 gap junctions become progressively confined to the transverse terminals of the cell, i.e. to the intercalated disks. Gap junctions and adhering junctions are frequently not closely adjacent in the neonate but become so with growing age (investigation for the first 6 years of life). It is presently still uncertain whether phosphorylation processes play a role in gap junction formation. However, a relation between phosphorylation of Cx43 and its insertion into the plasma membrane has been described [Musil et al., 1990b]. Furthermore, a correlation between the formation of functional gap junctions and expression of a cell adhesion molecule (L-CAM) and E-cadherin was reported [Mege et al., 1988; Musil et al., 1990b]. Berthould et al. [1993] showed that a reduction in extracellular calcium led to a loss of intercellular contact associated with a decrease in gap junctional intercellular communication as seen from reduced dye coupling and decreased anti-Cx43 immunofluorescence. Restoration of the extracellular calcium concentration resulted in reapparation of Cx43 immunoreactivity indicating the crucial role of calcium for the gap junction formation process. This was, however, unrelated to changes in the phosphorylation of Cx43. Stimulation of PKC with the phorbol ester TPA over periods longer than 15 min decreased immunolabeling at appositional membranes and increased cytoplasmic labelling. The extracellular loops E1 and E2 seem to determine the formation of the gap junctional channel. This was inferred from a study by Warner et al. [1995] using synthetic peptide analogues to extracellular loop segments in order to disturb the establishment of cellular coupling in pairs of embryonic chick heart myoballs expressing Cx43 and Cx32. Peptides resembling conserved motives from extracellular loops E1 and E2 delayed gap junction formation in micromolar concentrations. The motives QPG and SHVR in loop E1 and SRPTEK in loop E2 were critical for gap junction formation (one letter code, see Appendix). Interestingly there was no synergism between the peptide analogue to E1 and the analogue to E2, which were both about equi-effective. This means that it is critical if the formation is disturbed in only one loop. The processes involved in gap junction formation and assembly are summarised in figure 18.

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Fig. 18. Synthesis, posttranslational modification and assembly of gap junctions.

Structurally it has been found that large gap junctions are surrounded by small gap junctions (0.5 lm in diameter containing 12–100 connexons) which are located in the plicate and interplicate region of the intercalated disk [Severs, 1990]. It has been suggested that some of these gap junctions contain newly formed connexons freshly inserted into the lipid bilayer. However, this has never been proved. Regarding the fate of gap junctions Mazet et al. [1985] suggested that gap junctions facing the extracellular surface of dissociated myocytes are progressively internalized and form cytoplasmic vesicles which migrate into the cell interior and are degraded by lysosomal enzymes. The endocytotic internalization process was also confirmed by Severs et al. [1989], but over a period of 15–22 h neither degradation nor synthesis of new gap junction was observed. They concluded degradation to be much slower than previously assumed. However, this is probably relevant for freshly isolated or disaggregated cells used in experimental research and may account for the

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well-known ‘run-down’ of intercellular coupling in the course of such experiments, but may have less relevance for the situation in the intact tissue in vivo. Degradation of gap junctions seems to involve removal of the gap junction from the plasma membrane by internalization of the entire gap junction within one of the adjacent cells [Larsen, 1983; Mazet et al., 1985]. The interdigitating process is pinched off and a double-wall vesicle is formed, which is finally degraded within a lysosome. Within a cell so-called ‘annular’ gap junctions have been seen representing circular profiles which are cross-sections of an interdigitation or vesicle. Although Cx43 can be degraded in both lysosomes and proteosomes Tadros et al. [1996] have recently shown evidence of a major proteolytic degradation of Cx43 in lysosomes in the heart. Chen et al. [1989] described so-called gap junction-associated vesicles (GJAVs) in mammalian atrial and ventricular muscle. These GJAVs were located in the extracellular space in close vicinity to the intercalated disks in the interstitial space near gap junctions associated with plicate segments, within some t-tubular profiles and between the layers of the basal lamina covering the nonjunctional membrane close to the interplicate segment. Negatively staining with La(NO3)3 revealed that these GJAVs contained laminar structures which have been identified as typical connexon arrays. Pairs of these GJAVs form typical junctional pentalaminar structures. It was suggested that GJAVs resemble reservoirs of Cx43 and connexons possibly involved in the formation and degradation of gap junctions. The authors suggested that they may represent extracellular tracks for myocyte cell processes helping to meet or to retract from the neighboring cells. However, further studies on this subject are required to fully exclude artefacts from preparation methods. What about the real turnover rate of gap junction proteins as the basis of changes in gap junction pattern in the course of cardiac disease? There are several reports on a considerably high and rapid turnover of connexins both in vivo and in vitro. Laird et al. [1991] determined the turnover and posttranslational modification of Cx43 in neonatal cardiomyocytes. After labelling with 35S-Met, immunoprecipitation with anti-Cx43 antibodies followed by SDS-PAGE and fluorography revealed a phosphorylated and a non-phosphorylated form of Cx43. In pulse-chase experiments the half-life of Cx43 was determined with 1–2 h, and, furthermore, the turnover rate of the phosphate groups was experimentally defined by the half-life of the protein, i.e. phosphate groups can remain with the protein throughout its whole life span. This means that, at least in neonatal cardiomyocytes, there is a rather rapid turnover of Cx43. Valiunas et al. [1997] determined a formation rate of 1.3 channels/min after bringing cells into contact. Similarly, a considerably rapid turnover has been observed for Cx43 in other cultured cells [Musil et al., 1990b]. In addition, a

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half-life of 1.9 h for Cx43 was demonstrated in pulse-chase experiments in cultured rat heart ventricular myocytes by Darrow et al. [1995]. In the same study the half-life for Cx45 was determined with 2.9 h, suggesting a rapid turnover for both connexin isoforms. With regard to phosphorylation of the connexins these authors observed various phosphorylations of Cx43 on serine and threonine residues producing multiple forms of the protein but only phosphoserine in Cx45 which exhibited substantially less heterogeneity of phosphorylation. The role of connexin phosphorylation is uncertain at present. However, it is likely that phosphorylation, especially different phosphorylation (at various sites as in Cx43), may control various aspects of gap junction metabolism and function. The findings of Darrow et al. [1996] suggest that there might be a precursor pool for Cx43 whereas Cx45 may be synthesized de novo (see below). A short half-life of proteins is often associated with proline, glutamic acid, serine and threonine-rich regions (so-called PEST-rich regions) [Rechsteiner, 1988]. Taking a close look at the amino acid sequence of Cx43 reveals that there is no classic PEST-rich region. However, residues 272–285 and 327–340 on the C terminus may resemble PEST-like regions which may account for the rapid turnover as suggested by Laird et al. [1991]. Regarding other connexins, the turnover of Cx32 and Cx26 in cultured liver cells has been determined to be in the order of only several hours [Traub et al., 1989] and thus to be similarly rapid. In former years it was believed that this rapid turnover in hepatocytes is the fastest turnover of connexins, but in the mean time it is generally assumed that the connexins are probably all subject to rapid turnover.

5.1 Regulation of Gap Junction Synthesis by Intracellular Mediators The synthesis of gap junctions can also be regulated. An increase in cAMP, for example, increases junctional conductance over several hours, which can be inhibited by blockers of the mRNA synthesis [Kessler et al., 1985] or protein synthesis [Azarnia et al., 1981; Kessler et al., 1985; Traub et al., 1987] indicating an increased synthesis of the gap junction protein to be involved in this kind of long-term regulation. Similarly, In’t Veld [1985] observed a rise in gap junctional particles between rat pancreatic B cells following a rise in intracellular cAMP. Interestingly, sequences corresponding to the cAMPresponse elements are close to the transcription start site of Cx32, so that it has been suggested that cAMP may enhance transcription of Cx32 [Miller et al., 1988]. Saéz et al. [1989] reported that cAMP delayed the uncoupling

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of gap junctions in rat hepatocytes. This was ascribed to a possible decrease in the removal of gap junction proteins from the plasma membrane, i.e. to a slowing of degradation. Recently, Darrow et al. [1996] showed an increased expression of Cx43 and Cx45 following dibutyryl-cAMP exposure of neonatal rat cardiomyocytes, which was accompanied by an increase in conduction velocity assessed by optically mapped action potential propagation using voltage-sensitive dyes. However, the molecular mechanisms of this enhanced expression appeared to be different for both connexins. After 1 mmol/l db-cAMP the authors observed an increase in Cx45 but not in Cx43 synthesis within a 2-hour interval. In contrast, 24-hour exposure to db-cAMP resulted in an increase in Cx43 mRNA but not Cx45 mRNA, normalized to the GAPDH transcript. This increase was not attributable to synthesis of new protein factors as was indicated by the insensitivity to cycloheximide treatment. It is uncertain whether the increased Cx43 mRNA levels are due to enhanced transcription or to stabilization of the transcripts. The selectivity of the effect might indicate the action of a specific promoter or enhancer sequence near the Cx43 gene. The lack of sensitivity to cycloheximide suggests a possible modification of proteins already existing, for example, by a change in the phosphorylation state as a signalling event in the cAMP cascade. From these results the authors concluded that Cx45 may be upregulated posttranscriptionally (no change in mRNA, but in protein synthesis). In conclusion, the increased total amount in Cx43 by immunoblotting and, parallel to it, the lack of change in the protein synthesis rate of Cx43 reported by the authors may possibly be indicative of a precursor pool of Cx43. In contrast to cAMP, a stimulation of PKC with phorbol esters (TPA) has been shown to play an important role in the downregulation of gap junctional coupling [Yancey et al., 1982]. Since reestablishment of intercellular coupling was not seen after wash out of TPA in the presence of the protein synthesis inhibitor, puromycin [Fitzgerald et al., 1983], the phorbol ester probably induces the elimination of junctional channels under these conditions. Thus, it might be possible that PKC is involved in the regulation of channel degradation. Obviously, there is some kind of cross-talk between PKC and cAMP in the modulation of intercellular coupling, since cAMP can inhibit the uncoupling effect of phorbol esters if cells are exposed to both agents from the start of the experiment [Kanno et al., 1984], but this protective effect can be abolished by the protein synthesis inhibitor cycloheximide [Enomoto et al., 1984] in Balb/c3T3 cells. At least tyrosine kinases seem to be involved in the regulation of connexin expression. As stated previously, tyrosine kinases are often linked to growth factor receptors. A possible involvement of tyrosine kinases in the regulation

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Fig. 19. The Cx43 promoter region. Partial analysis of the human Cx43 gene extending from –360 to the site of fusion where De Leon et al. [1994] inserted the luciferase reporter gene at position +143. The transcription start site is numbered –1 [redrawn from De Leon et al., 1994]. The TATA box and the putative AP-1-binding sequence are underlined.

of gap junctional coupling seems to be reasonable in the light of Loewenstein’s [1968] hypothesis on the role of gap junctional communication in cellular proliferation. It was reported by Pepper and Meda [1992] that basic FGF exposure of microvascular endothelial cells leads to increased expression of Cx43. In contrast to these results, Doble et al. [1996] found decreased metabolic coupling in cardiac myocytes in response to FGF-2, the FGF which is believed to participate short- and long-term on cardiac responses to injury, within 30 min. However, in these experiments FGF-2 did not affect Cx43 mRNA or protein synthesis. With regard to the intracellular distribution of Cx43, FGF-2 exposure decreased immunofluorescence-staining intensity at sites of intermyocyte contact and induced phosphorylation of Cx43 in serine and tyrosine residues. In other cells (cardiac fibroblasts), however, the same authors demonstrated enhanced intercellular coupling by induction of Cx43 accumulation [Doble and Kardami, 1995].

5.2 Molecular Biology/Expression of Gap Junctions At present, only little is known on the molecular genetics of the connexins. Fishman et al. [1990, 1991a–c] isolated the entire gene encoding Cx43 including about 5,000 base pairs of 5-flanking sequence, a region which may determine the transcriptional activity of the gene. Regarding the molecular biology of the gap junction channel, Cx43 has been investigated in detail and the promoter

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region has been analyzed [De Leon et al., 1994] by construction of chimeric luciferase reporter genes containing nested deletions from the human Cx43 gene (–2,400 to –50 base pairs, position relative to the transcription initiation). The transcriptional activity of the chimeric genes was assayed in several cell types. High levels of luciferase activity required at least 175 base pairs of 5flanking sequence, whereas constructs which included 2,400 base pairs of the upstream sequence increased activity twofold in vivo but failed to increase activity in vitro. It was concluded from the experiments with several chimeric constructs that the proximal promoter may also confer tissue specificity. These studies begin to characterize the cis-acting elements of the Cx43 gene. These regulate the strength and specificity of transcription. The promoter includes a TATA box (TTTTAAAA) and a putative AP-1-binding site (TGAGTCA). The full sequence of the Cx43 promotor region is given in figure 19 according to De Leon et al. [1994]. Regulation of Cx40 expression has been suggested to be somewhat different from that of the other connexins: Darrow et al. [1995] suggested from their turn-over experiments a translational regulatory mechanism for Cx40 since they observed Cx40 mRNA, i.e. transcription took place, but they could not find the protein suggesting that either the protein is not translated or after translation rapidly degraded.

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6

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Gap Junctions in Cardiac Disease

In this chapter changes in the distribution of gap junctions within the myocardial tissue, alterations of the distribution of special isoforms in the course of heart disease are described. Thus, changes in gap junction pattern for Cx43 and for Cx40 in the border zone of a chronic infarction are pointed out. Changes with growing age and in the course of heart failure are discussed as well.

6.1 Gap Junctions in Acute Cardiac Disease One of the most intriguing problems in cardiovascular medicine is the acute myocardial ischemia and infarction, which often leads to lethal arrhythmia. There are many factors involved in the pathophysiology of cardiac ischemia and arrhythmogenesis [for review see, Janse and Wit, 1989; Katz, 1992]. The lack in oxygen and glucose supply leads to a loss of intracellular ATP and consequently to a failure of the Na+/K+-ATPase [Coronel, 1988; Gettes, 1987, Rosen et al., 1987]. This results in depolarization of the membrane potential [Kle´ber et al., 1978, Kramer and Corr, 1984] and influx of calcium which is further enhanced by reduced calcium elimination via Ca2+-ATPases or by exchange of the accumulating sodium against calcium. The mechanisms of calcium overload are complex and currently under investigation. In addition, K+ channels open and the extracellular [K+] rises to values of about 30 mmol/l or even more [Hirche et al,. 1980] in the interstitium of the tissue. This is enhanced by the opening of IK.ATP channels if ATP is reduced to very low concentrations [Furukawa et al., 1991; Wilde et al., 1990]. It is not certain at present whether other factors may also contribute to this K+-efflux. However, this K+-efflux is of pathophysiological importance since it can lead to further depolarization, to depolarization of surviving Purkinje strands [Lazzara and Scherlag, 1984], to the so-called injury current which can depolarize other fibers [Janse et al., 1980; Janse and van Capelle, 1982] and to action potential shortening. Reduction in action potential duration and conduction velocity results in a decrease in the local wave length which, thus, becomes heterogeneous with regard to its local distribution between ischemic and nonischemic heart. Differences in wave length are known to cause reentrant arrhythmias [Allessie et al.,1973; Krinsky, 1981]. Slowing of conduction is assumed to be

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a key factor in the initiation of reentrant arrhythmia [Janse and Wit, 1989; Pogwizd and Corr, 1987, 1990]. The situation becomes much more complex if the biochemical alterations, hemodynamics (especially the hypotension resulting from pump failure) and sympathetic activation with release of noradrenaline and subsequent tachycardia are also taken into account. Among biochemical alterations the accumulation of long-chain acylcarnitines has been discussed to play a role in gap junctional uncoupling. Normally, long-chain fatty acids can be transported into the mitochondrium by binding to carnitine (via acylcarnitine transferase I), passing the mitochondrial membrane as longchain acylcarnitine. The acyl group is then transferred to intramitochondrial CoA. In the course of ischemia this mechanism is altered and long-chain acylcarnitines accumulate within 2 min after the onset of ischemia in vivo [DaTorre et al., 1991]. Interestingly, there was a sevenfold accumulation of acylcarnitine in the junctional sarcolemma as compared to the nonjunctional regions [Wu et al., 1993]. Exogenous application of long-chain acylcarnitines resulted in rapid onset of cell-to-cell uncoupling [Wu et al., 1993]. Inhibition of accumulation of long-chain acylcarnitines significantly reduced the incidence of arrhythmia induced by ischemia in vivo [Corr et al., 1989]. In addition, Purkinje fibers exposed to lysophosphatidylcholines, which may be the case in subendocardium adjacent to ischemic myocardium, have been shown to generate early after-depolarizations [Arnsdorff and Sawicki, 1981]. Lysophosphatidylglycerides in combination with acidosis and elevated [K+] can induce delayed after-depolarizations and triggered activity in isolated Purkinje fibers [Pogwizd et al., 1986]. However, an enhanced extracellular potassium concentration and depolarization of the fibers besides the other factors lead to a reduced sodium channel availability, to a reduced maximum depolarization velocity, shortened action potentials and to a slowing of conduction. These changes result in an alteration in the activation patterns [Dhein et al., 1994] and an increase in dispersion of action potential duration, which is even more pronounced in the presence of neutrophilic leukocytes [Dhein et al., 1995a]. There are two forms of arrhythmia in acute myocardial ischemia. Type1a arrhythmias occur 2–10 min after the onset of ischemia with a peak at 5–6 min. These arrhythmias are often of the reentrant type and are caused by diastolic bridging (details see chapter 1). It is also possible that premature ventricular depolarizations occur in this phase and initiate reentry. Besides these, type-1b arrhythmia can occur at 12–30 min after the onset of ischemia with a peak at 15–20 min. These type-1b arrhythmias are either due to a partial recovery of the cell excitability (partial recovery of dU/dt and of the action potential duration), which may be ascribed to the release of catecholamines [for review see, Janse and Wit, 1989] or are due to gap junctional

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uncoupling and disturbed intercellular communication. From the considerations in chapter 1, it may be concluded that a partial recovery of excitability in concert with gap junctional uncoupling may cause inhomogeneities in the passive electrical properties of the tissue which may favor reentrant circuits, although it is yet uncertain whether reentry is the underlying mechanism of 1b arrhythmia (perhaps reentry in the ventricular wall outside the subepicardium) or other mechanisms, for example abnormal automaticity. What is the role of the gap junctions? Which of these changes may alter gap junctional gating? The loss of ATP, the fall in intracellular pH resulting from anaerobic glycolysis, the calcium overload, the rise in pCO2, the sodium overload of the fibers, fatty acids released in the ischemic tissue (see chapter 7), accumulation of long-chain acylcarnitines, leukotrienes from activated leukocytes, potential gradients between depolarized (ischemic) and normal tissue, all these changes will, as outlined in the previous chapters, result in gap junctional uncoupling. It is difficult or not possible to ascribe this effect to only one or two of these factors since they work in concert and cannot be separated from each other. But, is there really any evidence that gap junctions uncouple in the course of ischemia? Wojtcak [1979] reported an increase in internal longitudinal resistance in cow ventricular muscle after hypoxia. Dhein et al. [1997b] found an increase in the coupling time (time between stimulus and the propagated action potential>stimulus-response delay) 12 min after inducing hypoxia with concomitant glucose-free superfusion in guinea-pig papillary muscles (figure 20). In a more sophisticated setup Kle´ber et al. [1987] investigated the effect of ischemia on the propagation velocity and on internal longitudinal resistance in perfused rabbit papillary muscles. These muscles were perfused via a canula inserted in the coronary artery supplying the papillary muscle. Ischemia was induced by perfusion stop. In this setting about 15 min after induction of ischemia uncoupling occurred. Thus, gap junction uncoupling probably is not among the earliest changes in the course of ischemia but in later phases, i.e. after ?12 min, gap junctional uncoupling can occur and contribute to the changes in cardiac excitation spreading. In addition to intercellular coupling, Dekker et al. [1996] investigated the changes in intracellular calcium concentration in the course of ischemia in perfused rabbit papillary muscles. With regard to the mechanism of uncoupling, these authors favored the hypothesis that ischemia leads to an increase in intracellular calcium which was observed after 12.6 min of ischemia and was considered the main trigger for uncoupling. In a similar setup, Yamada et al. [1994] demonstrated that the accumulation of long-chain acylcarnitines contributed to cellular uncoupling in the course of ischemia and was delayed by inhibition of acylcarnitine transferase I. Interes-

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Fig. 20. Stimulus-response interval in guinea pig papillary muscle under normoxic and hypoxic conditions. Hypoxia was concomitted with glucose-free superfusion. Note the significant increase in the stimulus-response interval after 12 minutes of hypoxia.

tingly, uncoupling occurred concomitantly with the secondary rise in extracellular potassium. This secondary rise was also delayed by inhibition of acyltransferase I with 10 lmol/l 2-(5-(4-chlorophenyl)-pentyl)-oxirane-2-carboxylate (POCA). Since it has been shown that long-chain acylcarnitines can elevate intracellular calcium [Fischbach et al., 1992; Me´szaros and Papano, 1990], although inhibiting the L-type calcium current [Wu and Corr, 1992], Yamada et al. [1994] concluded from their experiments that long-chain acylcarnitine-induced uncoupling is due to an effect of the substance per se on gap junctional conductance and secondary to a rise in intracellular calcium. However, as outlined above, the alterations occurring in the course of ischemia, especially in the in vivo situation, are so complex that it may be difficult to ascribe a phenomenon such as cellular uncoupling to only a single factor. Since other factors occurring in ischemia can also contribute to uncoupling this phenomenon may be a multicausal rather than monocausal process including a rise in intracellular calcium [De Mello, 1975; Maurer and Weingart, 1987; Noma and Tsuboi, 1985], intracellular protons [Noma and Tsuboi, 1985], long-chain acylcarnitines accumulating in the junctional sarcolemma during hypoxia [Wu et al., 1993; Yamada et al., 1994] and reduced ATP content [Sugiura et al., 1990]. In vivo the situation may be even more complicated by

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the presence of activated leukocytes releasing lipoxygenase metabolites which have been suggested to be involved in gap junctional uncoupling and worsening of arrhythmogenesis [Dhein et al., 1995a, b; Gottwald et al. 1997; Massey et al., 1992]. In an interesting study Kieval et al. [1992] investigated pairs of cardiomyocytes isolated from rabbit hearts which had previously undergone global normothermic ischemia followed by 30-min of reperfusion in a Langendorff setup in comparison to cells isolated from hearts which were either perfused according to the Langendorff technique for 75 min (without ischemia) or isolated directly after removal of the heart without Langendorff perfusion. In all three groups of cells the action potential characteristics were normal. Mean Gj was also almost normal in all three groups but was significantly more widely distributed in the postischemic group with a greater population of cells exhibiting only poor communication. The authors thus concluded that postischemic myocytes resemble a heterogeneous population with regard to cellular coupling. Ultrastructural changes also occur in the course of acute ischemia. Ashraf and Halverson [1978] and McCallister et al. [1979] observed alterations in the gap junctional membranes after 20–30 min of ischemia. Hypoxia lasting for longer than 30 min induced loss of lipid aisles and in consequence a condensation of connexons in perfusion-fixed rat hearts with a subsequent rapid crystalline densely packed pattern for the next 10 min, i.e. after 40 min of hypoxia [De Mazie`re and Scheuermann, 1990]. At that time widespread cell damage became obvious and researchers speculated that the crystalline gap junction pattern may be associated with cell injury becoming irreversible. However, even very early in ischemia, i.e. 5 min after induction of ischemia, ultrastructural changes have been identified. Frank et al. [1987] suggested that rearrangement of sarcolemmal P-face particles may occur after 5 min and may resemble an unspecific response to alterations in membrane fluidity accompaning ischemia. However, the gap junctional surface density, i.e. the gap junction profile lengths per unit myocyte sectional area, is not altered at the onset of uncoupling at 30 min of hypoxia, although a reduction in the P-face center-to-center distance in freezefracture replicas has been observed. Since this reduction in P-face particles also occurs before the onset of uncoupling, it is considered not to play a primary role in the process of uncoupling [Hoyt et al., 1990; Peters, 1995]. However, it should be taken into account that other elements of the cytoskeleton are also important for the assembly of gap junctions as pointed out in the previous chapter and, thus, should also be investigated in the course of ischemia and infarction in order to find out the primary processes of uncoupling. What are the consequences of gap junctional uncoupling? Is it a benefit or a risk, or even both? Gap junctional uncoupling on the one hand will lead

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to electrical and metabolic isolation of the ischemic tissue. If between this tissue and the surrounding cells differences in action potential duration exist, these will be enhanced since coupling of the cells would smooth these differences as described by Dhein et al. [1994] and as suggested from computer simulations by Mu¨ller and Dhein [1993] and Lesh et al. [1989]. Such enhanced differences in action potential duration, mean enhanced dispersion, which is considered a risk factor for the occurrence of reentrant arrhythmia [Han and Moe, 1964; Kuo et al., 1983]. In addition, uncoupling also means slowing of conduction, which has also been considered a key factor in the initiation of reentrant arrhythmia [Janse and Wit, 1989; Pogwizd and Corr, 1987, 1990]. Uncoupling would also alter the activation pathways which may result in fractionation of the activation wavefronts and thereby lead to arrhythmia. On the other hand, isolation of the ischemic tissue will protect the surrounding tissue from the depolarizing influence which might induce arrhythmia via depolarization of Purkinje fibers. In addition this uncoupling may provide some kind of energy-saving effect for the tissue since the ischemic tissue is no longer activated and will, thus, stop contracting thereby reducing energy consumption. Thus, both beneficial and disadvantageous effects can result from gap junctional uncoupling. It probably depends on the local spatial distribution of the electrophysiological changes with regard to the microanatomy whether arrhythmia occurs resulting from altered pathways of excitation or not. However, the occurrence of late phase arrhythmias have been suggested to be related to gap junctional uncoupling [Dekker et al., 1996]. Another acute disturbance of the heart is acute arrhythmia. How do gap junctions behave in acute arrhythmia? One could imagine that acute tachycardic arrhythmias are concommitted by an increase in intracellular calcium and sodium as suggested [Bredikis et al., 1981] and possibly by a fall in intracellular ATP both possibly leading to uncoupling. In order to clarify that question Bredikis et al. [1981] submitted rabbit atrial muscles to high-frequency stimulation (10–15 Hz) for 15 min and measured the input resistance. They found an intercellular uncoupling in response to the rapid pacing with enhanced input resistance which recovered within 20–60 min after cessation of the rapid pacing. This tachycardia-induced increase was insensitive to treatment with atropine, propranolol or phentolamine. It is tempting to speculate that such an uncoupling induced by rapid heart rate may be an endogenous antiarrhythmic mechanism like some kind of a ‘selfdefibrillation’ mechanism, although this has not been shown unequivocally in controlled experiments. On the other hand, such uncoupling may also worsen the situation and provoke a change in the type of arrhythmia by altering the excitation pathways.

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6.2 Gap Junctions in Chronic Ischemic Cardiac Disease One of the most important chronic alterations in the heart is the chronic phase after myocardial infarction. The postinfarction period is known to be associated with an increased risk for sudden cardiac death and for the occurrence of cardiac arrhythmia. Changes in conduction properties have been identified [Dillon et al., 1988], although the cells exhibit normal or near normal action potential characteristics [Wit and Janse, 1992]. Thus, cellular electrophysiology does not explain the complete pathophysiology of the arrhythmogenic substrate. Thus, other factors, for example structural changes and passive electrical properties, have to be taken into account. Many factors contribute to this high risk of arrhythmia, especially the structural changes in the geometry of the tissue network. After infarction local contractility changes and the necrosis zone is replaced by connective tissue. FGF-2 can be released from cardiomyocytes during contraction and after stimulation with catecholamines. This factor is upregulated in response to myocardial damage [Doble and Kardami, 1995; Doble et al., 1996] and can decrease intercellular dye coupling. It induces Cx43 phosphorylation on serine residues, tyrosine phosphorylation and a masking of Cx43 epitopes in cardiomyocytes, whereas in fibroblasts coupling was found to be increased in response to FGF-2 (Doble and Kardami, 1995]. However, presently it is unclear whether this factor affects adult as well as neonatal cardiomyocytes. Myocardial injury which has been reported to cause increases in local FGF-2 [Kardami, 1990; Padua et al., 1993] could thus affect the intercellular coupling of the non-injured myocytes near the lesion. It is tempting to speculate that these changes might somehow be linked to the arrhythmias observed after myocardial infarction originating from abnormal conduction of activation in the vicinity of scar areas [Saffitz et al., 1992]. Experiments have been performed indicating that slowed anisotropic conduction may exist beyond the immediate interface with the infarct [Dillon et al., 1988]. However, what are the changes in gap junction distribution observed after myocardial infarction? Two major abnormalities regarding gap junction distribution have been observed in ischemic heart disease using laser-scanning confocal microscopy of anti-Cx43-stained specimens: (1) loss of the common ordered (polarized) distribution of the gap junctions, which was found predominantly in the border zone adjacent to infarct scars, and (2) reduction in the quantity of Cx43 gap junctions in areas distant from the infarct zone [Severs, 1994a, b]. This and other factors may result in a heterogeneous anisotropic conduction and locally reduced conduction velocity forming a proarrhythmic substrate. The active properties of cells and resting membrane potential can be quite normal in the presence of manifest cardiac arrhythmia,

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so that one can conclude that possibly the passive electrical properties may be of importance [Dillon et al., 1988; Spach et al., 1988; Ursell et al., 1985]. In the light of today’s research gap junctions are one of the most important determinants of these passive conduction properties [Peters et al., 1993; Saffitz et al., 1992]. In hearts of patients suffering from end-stage ischemic heart disease and in biopsies from patients 3 months after myocardial infarction, it was found that the gap junction distribution in histologically normal areas is almost normal. In contrast, within the border zone of healed infarcts (some hundred micrometers from the infarct scar) the pattern of gap junction distribution is disturbed with a wide dispersion of the gap junctions over the whole cell surface instead of being confined to the intercalated disks at the cell poles. These border zone myocytes also exhibit substantial heterogeneity with regard to orientation and ultrastructure, and sometimes a disorganization of the intercalated disks. Myocytes were observed which communicated via cell processes with gap junctions in the absence of fasciae adherentes. Besides these changes, annular gap junction profiles were found indicating a possible internalization of gap junctions. Between all these cells normal cells also occur [Severs, 1994a, b]. Following experimental infarction in the dog heart (10 weeks after occlusion of the left anterior descending coronary artery) [Luke and Saffitz, 1991], a more diffuse interstitial fibrosis was observed which was associated with a reduction in the number of gap junctions per unit length of disk membrane and decreased gap junction size of long gap junctions at the transverse section planes. A selective reduction in the larger gap junctions which are normally found at the circumference of the intercalated disk (this arrangement is thought to facilitate an efficient intercellular current transfer) [Green and Severs, 1993] was found so that a decrease resulted in the proportion of total gap junction in the interplicate segments of the intercalated disk. The number of cells to which a cardiomyocyte is connected was reduced from 11.2 in control tissue to 6.5 in the fibrotic infarct border zone. Intercalated disk zones were less clearly defined, and groups of junctions maintaining the intercellular coupling were displaced. In addition, a reduction in the frequency of intercalated disks of the side branches of the cells was seen, which provide side-to-side intercellular contacts [Luke and Saffitz, 1991]. They found a reduction in connections of cells in primarily side-to-side apposition by 75%, while connection of end-toend apposed cells were reduced by only 22%. This should result in a disproportionate increase in resistivity in the transverse direction thus enhancing anisotropy, potentially contributing to the development of reentrant arrhythmia. Especially such side-to-side contacts are necessary for homogeneous wavefront propagation as was demonstrated in neonatal rat heart cell cultures, which were grown in a patterned structure [Fast and Kle´ber, 1993].

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Thus, it can be hypothesized that these changes in gap junction distribution contribute to the alterations in activation pattern associated with chronic myocardial infarction and to the enhanced arrhythmogeneity, e.g. to reentrant arrhythmias originating in the border zone of healed infarcts. However, this is not the only factor since, for example, changes in the geometry by incorporation of connective tissue will also alter the pathways of excitation and will cause inhomogeneity of anisotropy. Besides this, surviving strands of Purkinje fibers within the infarcted area can act as arrhythmogenic foci, or surviving ‘peninsulas’ of myocytes can form excitable bridges from one side of the infarcted zone to the other, thereby connecting two parts which otherwise would be isolated from each other [Factor et al., 1978]. In patients with triple-vessel disease and recurrently ischemic myocardium undergoing aortocoronary bypass operation, the gap junction surface area per unit cell volume was reduced by 47% (0.0027 versus 0.0051 lm2/lm3) [Peters, 1995; Peters et al., 1993]. Taken together these results indicate that patterns of electrical coupling and electrical continuity may change between the degenerated infarct zone and the ventricular myocardium adjacent to this area [Smith et al., 1991]. However, there is no widespread derangement in gap junction organization although there may be quantitative alterations in expression in the noninfarcted myocardium of the ischemic heart. An interesting question is: what happens to the coronary vessels? Do they also undergo alterations in cellular coupling in the course of atherosclerosis? Blackburn et al. [1995] investigated these questions in atherosclerotic lesions representing different stages of the disease, which were obtained from coronary arteries of hearts removed from patients undergoing cardiac transplantation. They investigated the artery segments after staining with a specific anti-Cx43 antibody for immunofluorescence using a laser scanning confocal microscope. The investigations were carried out with a double-labeling technique using a second cell-specific antibody. They found a colocalization of Cx43 with smooth muscle cells but not with macrophages, and confirmed this result by electron microscopy. In addition, regions of intimal thickening and early atherosclerotic lesions exhibited increased Cx43 expression between the smooth muscle cells, most prominent in regions of intimal thickening (?10-fold increase). The quantity of Cx43-positive gap junctions was lower in early atheromatic lesions than in regions with intimal thickening but was higher than in normal vessels. With further progression of the disease the Cx43 expression was found to be progressively reduced from enhanced levels in early and earliest stages towards decreased levels (as compared to undiseased vessels) in the most advanced atheromatous lesions. With this development the distribution of the gap junctions changed and they became more patchy with larger diameters of the individual junctions.

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6.3 Gap Junctions in Heart Failure Another cardiac disease often associated with cardiac arrhythmias is heart failure. Many factors including high catecholamine levels, dilated tissue geometry, changes in the b-adrenoceptor population, impairment of the regulation of the intracellular (diastolic) calcium concentration, possibly enhanced endothelin levels and many more contribute to altered cardiac function and make the heart more prone to arrhythmia. However, the question was whether, in addition to the well-known structural changes, gap junction alterations may also partially form the arrhythmogenic substrate. Thus, researchers were interested in whether in the course of heart failure gap junctional alterations may occur. In patients suffering from heart failure due to ischemic cardiomyopathy Severs [1994a, b] described two main alterations (1) changes in the normal spatial distribution of gap junctions at the border zone of healed infarcts, and (2) a reduction in the quantity of Cx43 in regions distant from infarct scars. In patients with cardiac hypertrophy from chronically pressure-loaded human left ventricles due to aortic valve stenosis, a general reduction in gap junction surface area per unit cell volume by about 40% (0.0031 versus 0.0051 lm2/ lm3) has been observed [Peters et al., 1993]. The gap junctions in the pathological tissue were larger than normal. The estimated gap junction content per cell was reduced [Peters et al., 1993]. A reduction by 30% in the gap junction surface per cell was observed [Peters, 1996]. However, the number of intercalated disks per myocyte and the mean density of packing of connexons at freeze-fracture in these hearts remained unchanged as compared to control hearts. In contrast to these findings, in guinea pigs with cardiac hypertrophy following renovascular hypertension Peters [1996] reported a substantial increase in Cx43 gap junction expression in the early phase. Gap junction surface density was increased by 45% per cell and by 30% per volume unit, which was contrary to the findings in hypertrophied human myocardium. However, the different pathophysiology should be taken into account. It might be speculated that factors like angiotensin II can alter cardiac growth and possibly the architecture of the tissue, although at present there is no experimental evidence for an alteration in connexin expression. In cardiomyopathic hamsters Luque et al. [1994] stained for Cx43 using confocal microscopy and found that some of the cardiomyocytes stain normally but others stain diffusely, with a pixel intensity distribution of the confocal images showing a 90% increase in the number of pixels and a 60% decrease in pixel intensity in the cardiomyopathic hearts as compared to control hearts. Thus, Cx43 seemed to be present in the cells but did not become localized on the membranes as in normal cells.

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Another important cardiovascular disease affecting the heart and often associated with the pathogenesis of heart failure is chronic hypertension. Such hearts exhibit complex structural changes and it has been asked whether there is also an alteration in the gap junction distribution. Thus, researchers have investigated hearts from hypertensive animals. In hearts from hypertensive rats Bastide et al. [1993] found a reduced expression of Cx43 but an enhanced expression of Cx40 involving myocytes from the working myocardium. Since both connexins possess different electrophysiological properties especially with regard to their sensitivity to transcellular voltage, the conductive properties of the tissue may thereby be altered and a proarrhythmic substrate may be formed. Taken together all these findings described in ischemic heart, heart failure and hypertension suggest that a reduction in Cx43 expression may be a general feature in heart disease and may contribute to the enhanced arrhythmogeneity in many cardiac disorders.

6.4 Gap Junctions in Arrhythmia Gap junctions have often been discussed to play an important role in initiation and maintenance of acute arrhythmia (see above and chapter 1). In summary, all states with reduced intracellular pH, enhanced intracellular calcium, reduced ATP levels, sodium overload, etc., can induce cellular uncoupling, leading to alterations in the activation pathways and the geometry of excitation and may thus induce arrhythmia (for a detailed discussion of the role of gap junctions in acute arrhythmia see chapter 1). The main effect of gap junctional uncoupling is to introduce or enhance discontinuities in the anisotropic tissue, thereby setting the stage for microreentry as discussed in chapter 1. Another effect of gap junctional uncoupling is the slowing of conduction which is also believed to be a prerequisite of reentrant arrhythmia. Besides acute arrhythmia, chronic arrhythmia is a common and important clinical problem and chronification of arrhythmia is only poorly understood, although this might be the basis for new antiarrhythmic treatments from a more pathophysiological viewpoint. One of the most intriguing questions is whether chronification of arrhythmia may be related to changes in the underlying tissue structure and geometry of cellular coupling. One of the most common forms of arrhythmia is chronic atrial fibrillation and it is well known that the longer this arrhythmia endures the harder it is to convert the heart to sinus rhythm. It has been hypothesized that at least this form of arrhythmia may induce structural changes thereby forming the arrhythmogenic substrate of a chronic arrhythmia.

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In chronic atrial fibrillation Van der Velden et al. [1996] recently showed changes in the Cx40 distribution pattern in goat atria with chronic fibrillation. In the goat model used, atrial fibrillation was induced via chronic high-frequency pacing and they observed that, after switching the stimulus off fibrillation persisted for a period depending on the time elapsed during high-frequency pacing. Additional results were found in the authors working group using a rat model of atrial fibrillation. As a particularity rat atrial cells are coupled via Cx43 (see previous chapters). Rat atria were bathed in an organ bath in saline solution (superfusion at 7 ml/min) for 24 h and stimulated at 10 Hz thereby inducing atrial fibrillation which persisted if stimulation was switched off. After this time the atria were frozen, processed for immunohistochemistry and stained for Cx43. It became obvious from the experiments that in atria excised and immediately frozen the typical distribution of Cx43 gap junctions at the borders of the cells (at the cell poles in longitudinal direction) could be observed. After 24 h in organ bath and beating at their spontaneous rate, this pattern was not changed, but after 24 h of atrial fibrillation the Cx43 distribution changed with a more disperse pattern without the typical polarization (figure 21). From these experiments in rats and goats described above it was concluded that chronic arrhythmia may represent a state in which the distribution pattern of gap junctions can be altered by a yet unknown mechansim. This change in the gap junction pattern may then form the basis for chronification of the arrhythmia. Taken together, all these findings point to a new understanding of the arrhythmogenic substrate as a more structural change reflecting the electrical network ‘heart’. As a main point, at least in some of the diseases alterations in the intercellular coupling, as a main determinant of the network, contribute to the formation of the arrhythmogenic substrate. Substances interfering either with the cellular coupling via gap junctions or with the regulation of expression and distribution of gap junction proteins may, thus, represent a new antiarrhythmic approach [Dhein and Tudyka, 1995].

6.5 Gap Junctions in Infective Heart Diseases Conduction disturbances are frequently found in acute and chronic Chagas disease. In cultures of neonatal rat hearts, changes in the gap junction distribution were studied to discover whether they were associated with the infection. In cultured cardiomyocytes infected with the unicellular parasite Trypanosoma cruzi responsible for Chagas disease, which is the most common cause of heart disease in South America, reduced gap junctional conductance

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a

b Fig. 21. Cx43 immunostaining of rat atria either native (a) or after 24 h of atrial fibrillation (b) [Dhein et al., 1997a].

and decreased expression of Cx43 at the junctional membranes have been observed using immunohistochemistry [Campos de Carvalho et al., 1992, 1994]. Similarly the lucifer yellow dye transfer between infected cells was significantly reduced. Synchronized spontaneous beating becomes less regular in infected cells. Interestingly, the total amount of Cx43 was found to be normal, but the intracellular distribution was altered with high levels of intracellular Cx43 and only little at the appositional membranes. In addition, the cellular electrophysiology is altered with shortened action potential, elevated intracellular resting calcium levels and altered response to a-adrenergic stimuli.

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The immunohistochemical findings were correlated with reduced intercellular coupling indicating a possible role of disturbed Cx43 expression and gap junction function in the pathogenesis of Chagas disease and the arrhythmias associated with that disease.

6.6 Gap Junctions in Defective Heart Development Genetic evidence has grown in the last years showing that connexins can play an important role in the regulation of specific development. It is now known that mutations in the gene encoding Cx32 causes X-linked CharcotMarie-Tooth disease [Bergoffen et al., 1993; Paul, 1995], a demyelinating peripheral neuropathy. It was tempting to speculate that at least some of the cardiac malformations may be linked to mutations in the connexin genes. Thus Reaume et al. [1995] investigated the role of mutations in the Cx43 gene on fetal development in a mouse model. They created a null mutation in mice in the Gja1 gene encoding Cx43. They generated a mutation in the Cx43 gene by homologous recombination in 129 strain R1 embryonic stem cells with a construct replacing main parts of the coding sequence with the neor gene which lacked a promotor. Homozygous cell lines were morphologically normal and differentiated to embryoid bodies exhibiting beating heart muscle and blood islets, but were completely lacking in Cx43. 82% of the cells were not dye coupled. From heterozygous cell lines germline chimeras were generated by injection into C57BL/6 blastocysts and the homozygous offspring from heterozygous crosses was analyzed. No viable homozygous offspring was found. The pups died shortly after birth with cyanosis and signs of failure of pulmonary gas exchange, although the lungs became expanded and breathing was initiated. As long as they were alive the pups exhibited labored breathing. No alterations in external morphology, gross anatomy were detected except an enlargement of the conus of the heart’s pulmonary outflow tract of the right ventricle. This region was filled with intraventricular septae dividing the outflow tract into separate or blind-ended chambers. Filling of the right ventricle with methylacrylate for corrosion casts revealed no passage of the resin to the pulmonary arteries. Reaume et al. [1995] concluded that these right ventricular dysplasias caused death in the neonates when the circulation changes and the lungs must become perfused. Other tissues normally expressing Cx43 as lungs, kidneys, brain and gut remained histologically normal. There was no increase or change in Cx40 and Cx45 mRNA levels in their experiments. In addition to these results, mutations in the COOH terminal of Cx43 may be underlying cardiac malformations in visceroatrial heterotaxia syndromes as

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reported by Britz-Cunningham et al. [1995]. These authors investigated Cx43 DNA from 25 normal subjects and 30 children suffering from various congenital heart diseases (including hypoplastic left and right heart syndromes, DiGeorge syndrome, septal defects, trisomy 13 and others) using the polymerase chain reaction. They expressed the mutant DNA in cell culture and investigated its effect on the regulation of intercellular communication. Within the children’s group 6 children were identified, all suffering from syndromes including complex heart malformations, with substitutions of one or more phosphorylatable serine or threonine residues. In the Cx43 DNA of 5 of these patients a substitution of proline for serine at position 364 was seen. If cells were transfected with the Ser364Pro mutant Cx43 they exhibited abnormalities in the regulation of intercellular communication. The authors concluded from their findings that mutations in the Cx43 gene leading to cell-to-cell communication deficiencies are associated with visceroatrial heterotaxia. Another group [Kass et al., 1994] reported on a possible involvement of Cx40 in cardiac malformations. They evaluated seven generations with an inherited conduction system defect and dilated cardiomyopathy. This defect exhibited autosomal dominant transmission and perturbed both AV conduction and cardiac contractility. Genome-wide linkage analysis revealed that polymorphic loci near the centromere region of chromosome 1 (chromosome 1p1–1q1) were linked to the disease locus with a maximum multipoint lod score of 13.2 in the interval between D1S305 and D1S176. The authors speculated from these results that mutations of Cx40 may result in conduction system disease and dilated cardiomyopathy, since both Cx37 and Cx40 have been mapped to chromosome 1pter–q12. Because Cx45 does not map to chromosome 1 and Cx37 to the distal p arm of chromosome 1, Cx40 remained as a candidate gene responsible for that disease. The role of gap junctions during development has been investigated further in preimplantation mouse embryos by Becker and Davies [1995]. Besides the normal expression pattern of gap junctions in these embryos, they studied the developmental and junctional organization in mice naturally exhibiting reduced cell-to-cell communication (DDK syndrome, defect located on chromosome 11, it has been sugested that in DDK syndrome the regulation of intracellular pH is disturbed leading to lower pHi which may uncouple cells) and in normal mice with experimentally altered gap junction permeability. In principle they found that gap junctional communication is critical for the maintenance of compaction and the differentiation of an organized epithelium in the embryo and, thus, for the preimplantation development. The DDK embryos appeared to be phenotypically normal until reaching the morula stage. Thereafter, cells start to decompact and the embryo dies before reaching the expanded blastocyst stage.

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6.7 Gap Junctions in the Aging Heart As discussed earlier in this book, it has been shown that there are considerable changes in the distribution and expression pattern of gap junctional channels with increasing age. However, these investigations primarily included the developing heart until maturity. It is presently not known whether the gap junction distribution or the expression of various types of connexins is changing with increasing age as far as senium is concerned. It will be difficult to elucidate this question because it is necessary to differentiate between age per se and heart disease which is worsened with age. Every even small or minimal infarction and increasing heart failure with age will cause changes in the distribution pattern, which are primarily related to the disease and not to age per se. However, it is well known that with increasing age microfibrosis is observed which in turn will seperate the fibers from each other and thereby enhance the degree of nonuniformity as discussed in the first chapter of this book. This is accompanied by a reduction in side-to-side connections [Spach and Dolber, 1986]. Thus, with increasing age the intercellular communication can be expected to be reduced probably due to structural changes in the tissue with deposition of collagenous fibers. Concomitant changes in the gap junction distribution are probably secondary to cardiac diseases, although at present an effect of age per se cannot be excluded.

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Pharmacological Interventions at Gap Junctions

In the previous chapters the role of gap junctions in cellular communication and in cardiac disease has been outlined. It became obvious that in many diseases the intercellular communication is reduced via gap junctional uncoupling or via reduced or altered expression of gap junctional channels. On this background a straightforward idea would be to simply enhance the gap junctional coupling by any agent. However, one has to consider that the uncoupling, as pointed out in the previous chapter, is confined to certain areas, the diseased areas for example, in the heart. In addition, the uncoupling also has some positive effects, for example a putative energy-saving effect in ischemia. What would happen if coupling were enhanced? There are at least two principal possibilities: on the one hand it can be imagined that if coupling were enhanced unselectively in the whole heart the uncoupled area would then be coupled to the normal tissue and would behave more like this, i.e. the ischemia-induced action potential shortening would be reduced. Electrical inactivation would be antagonized and energy consumption would be enhanced. Since the channel is also permeable to small molecules it can be anticipated that molecules like ATP can flow to the ischemic zone and will be degraded there thus enhancing the ATP depletion in an ischemic setting. Metabolites and ions from an ischemic zone (e.g. lactate, H+, Ca2+, K+) would be able to diffuse to the nonischemic zone possibly exerting unfavorable effects there. Within the undiseased zone activation patterns probably may change. The transition zone between altered and normal electrophysiological behavior may become broadened. On the other hand arrhythmia due to uncoupling may be prevented. If coupling is enhanced selectively in the previously uncoupled area only within that area cellular uncoupling would be antagonized, which means that the surrounding tissue would not be affected in the way described above. There might be a similar effect in the close border zone between diseased and normal tissue, but the effect would be confined to that zone. Inhomogeneities within the diseased zone would be smoothened whereas the normal tissue behavior would probably be less affected. This could especially smooth differences in action potential duration and thereby prevent, in some situations, from reentrant arrhythmia, since this is often related to differences in action potential duration, to dispersion. Another important factor in the initiation of reentry

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is the slowing of conduction velocity [Janse and Wit, 1989; Pogwizd and Corr, 1987, 1990], which would be favored by cellular uncoupling and prevented at least in part by improving intercellular communication. Thus, enhancing intercellular coupling may exert a prophylactic effect against arrhythmia if arrhythmia is due to uncoupling. However, if the coupling effect is unselective, it would probably postpone an impairing effect as discussed above for ischemia. From this theoretical point of view selective couplingenhancing effects on the previously uncoupled tissue would be desirable rather than unselective. In contrast to these considerations, another strategy to follow may be the uncoupling strategy. In certain situations it might be favorable to cut out a part of the tissue. In ischemia, it might be interesting to investigate whether a full direct uncoupling of the ischemic zone might exert a protective effect during reperfusion due to energy saving. However, a hardly achievable prerequisite would be a selective effect on the ischemic zone. Otherwise, uncoupling in the whole organ will probably make the heart more prone to arrhythmia as outlined in chapter 1. For example Rohr et al. [1997] showed that, in discontinuous tissue under certain conditions (if there is a pronounced mismatch between the current source which was represented by strands of cultured cells of 55 lm width in their experiments and the current sink being represented by the expansion of the strand to a rectangular monolayer), failure of anterograde activation from the small current source to the large current load occurred, whereas successful retrograde activation was seen in the opposite direction. Application of an uncoupling agent (palmitoleic acid) transiently led to successful anterograde propagation of electrical activation in the region of unidirectional block. While improving intercellular coupling may exert prophylactic antiarrhythmic effects under certain conditions, in acute manifest arrhythmia a reduction in intercellular coupling may stop the arrhythmia by slowing the velocity on the reentrant pathway so that wavelength and anatomic reentrant path length do not fit each other any longer, a prerequisite for reentry suggested previously by Krinsky [1981]. However, it should be considered that in both cases one has to distinguish whether a substance uncouples or couples the whole tissue or only those parts with altered intercellular communication. Thus, the question arises: what is presently known about the pharmacology of gap junction channels? In the following a survey is given of the substances which have been found to alter intercellular coupling. First drugs will be considered which uncouple gap junctions. A number of lipophilic compounds have been described to reduce gap junctional coupling. These substances include alcohols like heptanol and octanol, saturated and unsaturated fatty acids, and alcohols and

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volatile anesthetics like halothane and ethrane (enflurane). Halothane is often used in double-cell voltage-clamp or dye-transfer experiments to uncouple cells in concentrations of about 1.5 mmol/l [Burt and Spray, 1989; Moreno et al., 1994a; Nedergaard et al., 1995]. Ethrane is effective in concentrations of about 4 mmol/l [Burt and Spray, 1989]. Octanol can uncouple embryonic chick ventricle cells [Veenstra and DeHaan, 1988] and adult rat ventricular myocytes [White et al., 1985]. It has been suggested that this effect may be caused by limiting the channels from opening to their largest configuration, i.e. by interference with the switching between various conductance states [Chen and DeHaan, 1993]. With regard to the mechanism there is no effect of octanol on single-channel conductance itself in neonatal rat cardiomyocytes [Burt and Spray, 1988b]. As octanol, heptanol also reduces gap junctional conductance [Bastide et al., 1995; Kimura et al., 1995; Ru¨disu¨li and Weingart, 1989]. For experimental approaches this may be interesting since after application of 3 mmol/l heptanol single-channel behavior can be observed. According to Ru¨disu¨li and Weingart [1989] the effect is fully reversible within 2 min after washout in their experimental system. The concentration-response curve revealed a steep S-shaped relationship with a threshold concentration of about 10Ö4 mol/l and the maximum effect at a concentration of about 10Ö3 mol/l heptanol. Kd was determined to be 0.16 mmol/l and the Hill coefficient z>2.3 for the equation Gj>(Kd)2/((Kd)2+[heptanol]2). Regarding the mechanism of action Haydon et al. [1984] and Niggli et al. [1989] suggest that heptanol acts by incorporation into the plasma membrane and the lipid bilayer. Ru¨disu¨li and Weingart [1989] concluded from their findings that heptanol uncoupled cardiac cells via impairment of the open probability po, the gap junction conductance gj being described by the equation gj>N · cj · po (N>number of channels). Expression, phosphorylation or localization of Cx43 are not altered by brief exposure (5–20 min) to 2 mmol/l heptanol [Kimura et al., 1995]. The mechanism of action of heptanol was further clarified by Bastiaanse et al. [1993], who showed that the uncoupling effect was based on a decrease in the fluidity of membranous cholesterol-rich domains. Gap junctions are embedded in such cholesterol-rich domains of the membrane. The unitary conductances were unaltered by heptanol, so that the authors concluded that heptanol decreases the open probability as already shown in a previous study by this group [Takens-Kwak et al., 1992]. However, some authors showed that heptanol and octanol can also inhibit the cardiac sodium current [Nelson and Makielski, 1990] and that general anesthetics like octanol and decanol can interfere with the cardiac Na+/Ca2+ exchange [Haworth et al., 1989] at concentrations below those required for gap junctional uncoupling. This action is considered to contribute to their well-known negative inotropic effect.

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Another group of lipophilic substances which can uncouple cardiac gap junctions comprises fatty acids and alcohols. However, it depends on the length of the acyl chain and on saturation of the carbon bonds whether the fatty acids uncouple or not. Burt et al. [1993] systematically investigated the influence of various saturated and unsaturated fatty acids on junctional coupling. They found saturated fatty alcohols with an acyl chain length of 7–12 but not higher, as well as unsaturated C18 cis 9 fatty alcohol to be effective in uncoupling neonatal rat heart cells. Saturated fatty acids with acyl groups of 10–14 carbons but not more and unsaturated cis 9 fatty acids with acyl chain lengths of 14–18 exhibited an uncoupling effect as well. Decanoic acid in concentrations of 2 mmol/l rapidly and fully reversibly uncoupled cardiac cells. As with heptanol no change in cj was observed so that the investigators concluded that the uncoupling effects were due to a reduction in the open probability rather than in cj. These drugs are supposed to incorporate in the lipid bilayer and to increase disorder in the interior of the membrane (C9–C18 region) [Goldstein, 1984; Gruber and Low, 1988; Klausner et al., 1980; Pringle et al., 1981] thus acting by their physical properties rather than by chemical interaction. All drugs listed above found to be effective uncouplers exhibit high rotational and lateral mobility in the bilayer. The cis 9 acyl chains require more space for rotation than the straight-chain analogues. The short-chain compounds including arachidonic acid (see below) incorporate in the exterior volume of the bilayer, whereas halothane dissolves in the interior of the membrane. Although diverse in structure these lipophilic agents share a common physical property: incorporation into the membrane, disordering its structure and inhibiting gap junctional channels [Burt et al., 1993]. Interestingly, decanoic acid and palmitoleic acid can uncouple heart cells without affecting other transmembrane channels contributing to the action potential [Burt et al., 1991]. Another important feature is the finding that multiple lipophiles have additive effects [Burt et al., 1993]. Cells, however, differ with regard to their sensitivity to these lipophilic compounds. Adult rat heart cells, for example, are relatively resistant to uncoupling by these lipophiles [Ovadia and Burt, 1991]. Neonatal rat heart cells and A7r5 cells, a neonatal rat aortic smooth muscle cell line, seem to be more sensitive. The underlying mechanisms for diverse tissue sensitivity remain to be elucidated. According to the mechanism of heptanol-induced uncoupling [Bastiaanse et al., 1993] it is tempting to speculate that the cells might differ in their cholesterol-rich domains. In addition, at present it is not clear whether there are differences in the sensitivity of various connexins. Oleic acid for example has been shown to differentially affect gap junctional coupling between neonatal rat cardiomyocytes and A7r5 cells: low concentrations of oleic acid (up to 1 lmol/l) reduced dye coupling in A7r5 cells by 50%, but higher

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concentrations had no further effect. In contrast, neonatal rat cardiomyocytes became uncoupled to zero levels in a linear fashion in concentrations ranging from 1 to 25 lmol/l [Hirschi et al., 1993]. Although often used for uncoupling, one should keep in mind that the effect of these lipophilic agents is a physical effect on membrane structure. This means that these drugs are valuable tools for investigation of single-channel behavior, but they are probably not suitable for inducing uncoupling in order to investigate effects of putative coupling agents on the overall conductance gj if these agents are supposed to act via some receptor-coupled regulatory mechanism. Another agent uncoupling cardiac gap junctions is arachidonic acid and some of its metabolites. Exposure to arachidonic acid produces uncoupling in neonatal rat cardiomyocytes [Schmilinsky-Fluri et al., 1990]. The concentration response curve analysis revealed a Kd of 4 lmol/l and a Hill coefficient of 0.75. The uncoupling was reversible gj returning back to 61% after 30 min washout and recovery could be accelerated by addition of bovine serum albumin to the bath solution (gj reaching 86% of the initial value after 10 min of washout). The effect was specific for arachidonic acid and could not be mimicked with analogues like arachidic acid (100 lmol/l) or arachidonamide (10 lmol/l). The single-channel conductance cj (mean cj>33.5 pS) was not affected by arachidonic acid at concentrations ranging from 1 to 100 lmol/l, so that the authors concluded that arachidonic acid might reduce the open probability of the channel. In contrast, 100 lmol/l arachidonic acid did not affect nonjunctional membrane current in these experiments. 100 lmol/l arachidonic acid induced uncoupling starting after 90 s, and after 2.5 min junctional current was no longer detectable. In addition to these findings, Massey et al. [1992] reported that the uncoupling effect of arachidonic acid was not only dose- but also time-dependent and that the dose-response curve could be shifted to the right by pretreatment with 2.5 lmol/l U70344A, a 5-lipoxygenase inhibitor, whereas pretreatment with the cyclooxygenase inhibitor indomethacin (100 lmol/l) had no effect on the arachidonic acid concentration-response curve. Complete uncoupling occurred at membrane concentrations of 3–4 mol%. Incorporation of arachidonic acid into the lipid bilayer was not affected by the inhibitors. Complete uncoupling was achieved with 20 lmol/l arachidonic acid within about 3.5 min, with 5 lmol/l within 4.5 min and with 2 lmol/l within 9.5 min. Inhibition of 5-lipoxygenase delayed this uncoupling. It was suggested by the authors that arachidonic acid is metabolized via lipoxygenase to metabolites which contribute to the uncoupling effect. However, lipoxygenase products like leukotrienes themselves have not been investigated directly. Thus, it remains unclear whether 5-HPETE, 5-HETE or the leukotrienes act as uncoupling agents and whether this is a receptor-mediated effect.

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Long-chain acylcarnitines which increase rapidly within minutes after the onset of ischemia or hypoxia can also uncouple cardiac muscle and reduce gap junctional conductance [DaTorre et al., 1991; Wu et al., 1993; Yamada et al., 1994] (for details see chapter 6). Regarding pharmacological interventions, it was interesting that inhibtion of acylcarnitine transferase I by 10 lmol/l POCA or by 100 lmol/l oxfenicine completely prevented the accumulation of long-chain acylcarnitines even within 40 min of ischemia in arterially perfused rabbit papillary muscles and delayed the onset of and progression of uncoupling and ischemic contracture [Yamada et al., 1994]. The inhibitors did not influence the loss of intracellular ATP or the initial rise in extracellular potassium, whereas the secondary rise in extracellular potassium, concomitant with cellular uncoupling, was delayed. Is there any physiological or pathophysiological role for these findings? Corr et al. [1984] found that ischemia enhances lipid metabolism and thereby leads to the liberation of fatty acids. Besides this, it has been outlined above and in the previous chapter that ischemia results in the accumulation of longchain acylcarnitines. According to the findings described above these fatty acids may incorporate into the plasma membrane, disorder the lipid bilayer surrounding the gap junctional channels and, thereby, reduce the open probability of the channel and contribute to cellular uncoupling during ischemia. Cytosolic levels of arachidonic acid have also been reported to be increased in response to hypoxia or ischemia [Chien et al., 1984]. The finding that arachidonic acid can reduce the conduction velocity of the action potential [Bayer and Fo¨rster, 1979; Szekeres et al., 1976] may reflect its uncoupling action on gj. Thus, the release of both fatty acids and arachidonic acid may contribute to the enhanced arrhythmogenesis during ischemia. Pharmacological approaches include the inhibition of release of arachidonic acid by inhibition of phospholipase A2 and the inhibition of acylcarnitine transferase I by POCA and oxfenicine, the latter of which has been shown to prevent or at least delay ischemia-induced uncoupling. There are at present no data available on the possible effects of inhibitors of arachidonic acid release on ischemia-induced uncoupling. Gap junctions can also be uncoupled by weak organic acids. Acetic acid for example has been shown to effectively uncouple gap junctions in crayfish septate axons lowering pHi to values around 6.2 [Peracchia, 1991a; Ramon et al., 1991]. The uncoupling effect exhibits rapid onset and reversibilty. Similarly, Nedergaard et al. [1995] reported on an uncoupling effect of 10 mmol/l lactic acid adjusting the extracellular pH to values ranging between 6.48 and 7.30 in Hanks’ buffered saline solutions. Lactic acid facilitates the intracellular acidification under these conditions [Nedergaard et al., 1991]. Propionic acid can also be used for uncoupling experiments as shown by Gottwald and

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Dhein [1997]. If considering the mechanisms of the induced uncoupling there are two possibilities: first, intracellular acidification may directly induce cellular uncoupling via the pHi-uncoupling effect; second, the organic acid may enter the cell in its undissociated form, then dissociate and the resulting H+ may be exchanged against Na+ via the type-1 Na/H-exchanger as shown by Gottwald and Dhein [1997], thus inducing an intracellular sodium overload and eventually a secondary rise in calcium. Another group of drugs influences the intracellular sodium and calcium concentrations thereby modulating gap junctional coupling. These drugs will be described in the following paragraph. Among the drugs often used in cardiology the digitalis glycosides have been shown to uncouple cardiac myocytes. Using a silicon oil chamber Weingart [1977] demonstrated in cow hearts that the exposure to 2 lmol/l ouabain for 90 min increased longitudinal resistance from 420 to 1,032 Xcm and concomitantly reduced the conduction velocity from 50 to 29 cm/s. The increase in longitudinal resistance was associated with an increase in diastolic tension suggesting a rise in intracellular calcium as the underlying mechanism. Similarly, De Mello [1976] observed an uncoupling effect of 0.68 lmol/l ouabain in Purkinje fibers. He suggested an increase in intracellular [Na+] and a secondary increase in intracellular [Ca2+] as the underlying mechanism. It is widely accepted that exposure to cardiac glycosides in at least toxic concentrations produces an increase in intracellular [Na+] via inhibition of the Na+/K+-ATPase [Hoffman and Bigger, 1985] thereby decreasing the transmembrane sodium gradient which causes a secondary rise in intracellular [Ca2+] via the impairment of the Na+/Ca2+exchange mechanism. Besides this an increase in the slow inward calcium current Isi has been described [Gilman et al., 1985]. Weingart and Maurer [1987] studied the effects of exposing guinea-pig ventricular cell pairs to 2 and 20 lmol/l strophanthidin. They found a dose- and time-dependent uncoupling effect of strophanthidin on nexal resistance. 2 lmol/l produced uncoupling after 20–25 min and 20 lmol/l after 10–15 min. This could be accelerated if the pulse frequency in these experiments was enhanced from 0.3 to 1.0 Hz. Nexus resistance was enhanced by 2 lmol/l strophanthidin from 19 to 295 MX in these experiments. Inhibiting the transmembrane calcium current Isi antagonized the uncoupling effect of the cardiac glycoside indicating that extra Ca2+ influx via Isi contributes to the uncoupling action. It can be imagined that these uncoupling effects of the cardiac glycosides may contribute to the arrhythmogenic risk associated with digitalis therapy and intoxication. Another compound increasing the intracellular sodium concentration is the aconitine, a drug found in monkshood (Aconitum napellus) which is one of the most toxic plants in middle Europe. In cardiac muscle the alkaloid

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causes a prolonged sodium current with slowed repolarization. It is used in experimental pharmacology to produce ventricular arrhythmia. Although not yet shown directly, it can be imagined that such drugs, opening the fast sodium current, will increase intracellular sodium load and uncouple the cells according to the findings of De Mello [1976] who found a rapid uncoupling after sodium injection. Regarding manipulation of the intracellular calcium concentration, caffeine has been used in experiments on intercellular communication. If cardiac cells are exposed to methylxanthines such as caffeine a phasic release of calcium from the sarcoplasmic reticulum can be observed [Chapman, 1979]. Maurer and Weingart [1987] investigated the effect of exposing adult guinea-pig cardiac ventricular cell pairs to 5–10 mmol/l caffeine. This intervention did not change the gap junction conductance. However, if caffeine was applied after reduction of extracellular Na+ (to 15 mmol/l, which reduced gj by 20%), caffeine induced a rapid (within 90 s) decrease in gj (74%). After decoupling canine Purkinje cells by injection of calcium De Mello [1975] found a slowed recovery in the presence of 6 mmol/l caffeine (extracellular). It is difficult at present to interpret these results from a pharmacological point of view. Experiments have to be performed on the influence of caffeine or other drugs altering intracellular calcium balance on intercellular coupling and arrhythmogenesis in previously uncoupled preparations (preferably with high calcium). It has been shown in crayfish septate axons that the uncoupling effect of halothane could be enhanced by coadministration of caffeine, which might give a partial explanation for arrhythmias in patients treated with methylxanthines, like theophylline, during halothane anesthesia [Peracchia, 1991b]. According to the calmodulin hypothesis [Peracchia, 1988], it can be anticipated that calmodulin antagonists should exhibit an influence on gap junctional coupling. Indeed, Peracchia et al. [1983] and Peracchia [1987] demonstrated that calmodulin inhibitors were able to prevent cell uncoupling. In crayfish septate axon for example electrical uncoupling could be inhibited by the calmodulin inhibitor W7 [Peracchia, 1987]. On this background systematic studies on the influence of calmodulin antagonists on gap junctional resistance in several models of uncoupling would be highly desirable. Regarding calcium, there is one study dealing with the effect of the calcium channel antagonist, verapamil, on gap junctional conductivity. In the acinus of the rat submandibular gland the uncoupling effect of the secretagogue acetylcholine as assessed in dye-coupling studies could be inhibited in the presence of 10 lmol/l verapamil [Kanno et al., 1993]. This is probably due to the antagonization of calcium influx. In control cells (without uncoupling by acetylcholine) verapamil did not influence cell coupling. Unfortunately, there

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are no data on the verapamil action on cardiac cells previously uncoupled by any calcium agonistic process available. Another possibility of influencing gap junctional coupling is the modulation of the activity of intracellular phosphatases and protein kinases using okadaic acid, staurosporine and phorbol esters. It has been outlined in the previous chapters that single-channel conductance of various connexins is regulated by phosphorylation and dephosphorylation processes. Thus, it can be expected that drugs inhibiting or stimulating phosphatases or protein kinases can alter gap junctional conductance. Moreno et al. [1994b] found that treatment of SKHep1 cells transfected with human Cx43 with okadaic acid (300 nmol/l), an inhibitor of phosphatases type 1 and 2A, changed the frequency distribution of unitary junctional conductance. Under the influence of okadaic acid a shift in the single-channel conductances from higher to lower conductance favoring a 60-pS conductance state was observed. Under these conditions the phosphorylation of human Cx43 was increased. The reverse effect could be expected if phosphorylation of Cx43 would be inhibited by an inhibitor of protein kinases. Such an inhibitor is staurosporine which inhibits PKC and cyclic nucleotide-dependent protein kinases. Moreno et al. [1994b] investigated the effect of 300 nmol/l staurosporine on singlechannel conductance and observed a decrease in the frequency of 60-pS events and an increase in 100-pS events. Thus, the unitary conductance can be modulated pharmacologically. However, since the global gap junctional conductance depends on single-channel conductance and open probability, the overall effect on coupling cannot be concluded from these experiments. Because PKC has been shown to increase gj [Kwak & Jongsma, 1996; Spray and Burt, 1990], it can be anticipated that staurosporine, as an inhibitor of this enzyme, may exhibit a decreasing effect on gj. On the contrary, the overall effect of okadaic acid might consist of an increase in gj. However, more experiments will be necessary for a final statement. In addition, it is possible to stimulate protein kinases directly by treatment with phorbol esters [Kwak and Jongsma, 1996; Moreno et al., 1994b; Mu¨nster and Weingart, 1993]. Mu¨nster and Weingart [1993] reported that exposure of neonatal rat heart cells to 100–160 nmol/l TPA, a stimulator of PKC, led to a rapid decrease in the gap junction conductance gj. The onset of uncoupling was observed 2–9 min after TPA application and maximum uncoupling within 2–4 min thereafter. They concluded that TPA may affect channel kinetics rather than the single-channel conductance cj. The TPA effect occurred only acutely; 24-hour exposure of the cells to TPA did not result in changes in gj attributable to downregulation of PKC. The TPA-induced change in gj can be prevented by pretreatment with the PKC inhibitor, staurosporine. In further experiments Mu¨nster and Weingart [1993] showed that the TPA effect depends

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on the free intracellular calcium concentration: at low intracellular calcium levels (18 nmol/l) the uncoupling TPA effect can be observed, whereas at higher levels (100 nmol/l) the effect becomes mitigated or is completely inhibited (160 nmol/l). This finding is somewhat contradictory since it is well known that PKC requires Ca2+ for its action. However, the calcium sensitivity of the various isoforms of PKC is different with PKC-b exhibiting substantial activity in the absence of calcium and an undefined isoform without calcium-sensitivity [Nishizuka, 1988]. In addition, the subcellular distribution of calcium under these conditions is not clear. At present, it is not possible to interpret this calcium-PKC inhibitor interaction on a mechanistic level. In addition, it is not clear what the effect of TPA or other phorbol esters on cellular coupling in intact tissue might be, since in intact cardiac tissue the resting intracellular calcium concentration ranges to about 150 nmol/l [Wier et al., 1987]. Other investigators observed an increase in gj in response to TPA [Kwak and Jongsma, 1996; Spray and Burt, 1990]. Kwak and Jongsma [1996] found an increase by 16×2% in gj in neonatal rat cardiomyocytes after application of 100 nmol/l TPA (intracellular calcium was buffered with 10 mmol/l EGTA in the pipette solution). TPA shifted the frequency distribution of unitary conductances cj to lower sizes. However, TPA decreased dye coupling in these and other experiments [Kwak et al., 1995a]. The uncoupling TPA effects can also be mimicked with synthetic diacylglycerol analogues such as 1-oleoyl-2-acetyl-glycerol [Mu¨nster and Weingart, 1993] in concentrations of 250 lmol/l, which also activates PKC. Lower concentrations are ineffective. Besides these approaches which act at intracellular enzymes, modulation of the autonomous nervous system by receptor agonists can alter gap junction conduction. The parasympathomimetic carbachol for example, a drug which acts at muscarinic and nicotinic acetylcholine receptors and is not susceptible to cholinesterases, raises intracellular cGMP and can thus be expected to decrease gap junctional coupling via cGMP-dependent protein kinase. TakensKwak and Jongsma [1992] investigated the influence of 100 lmol/l carbachol on gj in cultured neonatal rat cardiomyocytes. In the whole cell technique carbachol exposure decreased gj by 20%. The frequency distribution of unitary currents was shifted by carbachol from 43 pS to a lower cj of about 21 pS in heptanol-uncoupled cells. The authors argued that a cGMP-dependent protein kinase phosphorylates the channel and thereby closes the 40- to 45-pS channels without affecting the other population of 20-pS channels. A similar decrease in gj was obtained using 1.5 mmol/l 8-bromo-cGMP. The carbachol effect was not seen in the perforated patch technique due probably to loss of an intracellular cytosolic phosphatase. In dye-coupling experiments Shibata et al. [1995] also observed reduced coupling in response to 100 lmol/l carbachol in cultured adult

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rat and guinea-pig cardiomyocytes. However, uncoupling occurred only in the presence of calcium whereas in the absence of calcium carbachol did not repress dye coupling between the cells. The uncoupling effect of carbachol has also been established in other cells, for example pancreatic acinar cells [Somogyi and Kolb, 1989] or rat submandibular gland [Kanno et al., 1993]. In the latter the effect of carbachol could be prevented by coadministration of atropine and could be mimicked with the cholinomimetic natural alkaloid pilocarpine. Sympathomimetics have also been studied with regard to their effects on gap junctional coupling. Epinephrine has been shown to increase the spread of electrotonic potentials during diastolic depolarization [De Mello, 1986b] in canine Purkinje fibers. This was interpreted as an effect of the rise in intracellular cAMP resulting from b-adrenoceptor stimulation and subsequent formation of cAMP by adenylate cyclase which then activates PKA. Details regarding the regulation by cAMP and by PKA have been described in chapter 4. Similarly, De Mello [1989] reported on an improvement in intercellular coupling by the b-adrenoceptor agonist isoproterenol in cardiac cell pairs. Thus, stimulation of b-adrenoceptors can be assumed to result in enhancement of intercellular coupling, at least in some preparations. However, on the basis of the findings of Kwak and Jongsma [1996] on a lack of the effect of PKA to alter gap junction conductance in rat cardiomyocytes, caution seems necessary and species variability or tissue variability seems to play an important role. In other cells, i.e. in rat submandibular gland, adrenaline (100 lmol/l) has been shown to decrease the percentage of dye-coupled cells [Kanno et al., 1993], whereas isoproterenol was ineffective, so that the authors concluded that the mechanism was transmitted via action on the a-adrenoceptors. This was supported since the adrenaline effect could be suppressed by coadministration of 10 lmol/l phenoxybenzamine. Similar to a b-adrenoceptor stimulation intracellular cAMP can be increased by inhibition of phosphodiesterase. Thus, in turtle retina cells, cAMP leads to uncoupling and this can be mimicked by stimulation of adenylate cyclase with forskolin and concomitant inhibition of phosphodiesterase by IBMX [Piccolino et al., 1984]. In cardiac cells inhibition of phosphodiesterase has been investigated using methylxanthine derivates [De Mello, 1989], resulting in an enhancement of intercellular coupling. It should be kept in mind that stimulation of a given protein kinase can increase or decrease gap junctional conductance depending on the tissue and species studied. Thus, generalizations should be avoided. Norepinephrine-dependent phosphorylation of connexins by PKC has been described in liver cells expressing the 27-kD gap junction protein [Takeda et al., 1989]. It can be assumed that in cardiac cells this would lead to the same effects as direct stimulation of PKC via phorbol esters. Indirect evidence

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for an improvement in cellular coupling by norepinephrine via a1-adrenoceptors has been found in isolated rabbit hearts perfused according to the Langendorff technique, exposed to increasing concentrations of norepinephrine in the absence and presence of the b-blocker, propranolol, and the a1-adrenoceptor antagonist, prazosine [Dhein et al., 1993a]. It became obvious in that study that norepinephrine decreased the dispersion of action potential duration measured at 256 ventricular electrodes after blockade of the b-adrenoceptors by propranolol. The effect could be suppressed by prazosine. Since the enhancement of the dispersion of action potential duration can be the result of cellular uncoupling [Lesh et al., 1989], the authors interpreted this action as a possible improvement in intercellular coupling. In addition to the modulators of the autonomic nervous system, angiotensin II was found to be effective in regulating cardiac gap junction conductance. In adult ventricular cell pairs De Mello [1992] observed a 55% reduction in gap junction conductance gj within 20 s following the administration of 1 lg/ml angiotensin II (approximately 0.9 lmol/l) to the bath solution. The angiotensin-II effect was reversible within 2.5–3 min. The concentration-response curve started at 10 nmol/l resulting in a decrease of 18%. The uncoupling angiotensin-II effect could be prevented by the PKC inhibitor staurosporine and could be suppressed by DuP 753 (70 lg/ml), an angiotensin receptor-blocking agent, whereas DuP 753 alone did not alter gj. In additional experiments De Mello [1992] investigated the influence of the angiotensinconverting enzyme inhibitor, enalapril (1 lg/ml). Enalapril was added to the bath solution and resulted in an increase of 106% within 4–5 min. The onset of the effect took 1.5 min, probably the time needed in vitro for ester hydrolysis of enalapril to MK-422, the active metabolite. The enalapril effect was dosedependent in the range from 0.25 to 1.25 lg/ml, reaching maximum effect at 1.0 lg/ml. In further investigations De Mello [1994] examined the possible existence of an intracellular renin-angiotensin system. In this study using adult rat heart cells intracellular dialysis of 10 nmol/l angiotensin I resulted in a decrease in gj of 76% within 7 min. This could be completely inhibited by intracellular dialysis of 1 nmol/l enalaprilat. Intracellular dialysis of angiotensin II led to a decrease in gj of 60% in 45 s and was sensitive to PKC inhibition. The author concluded that there is an intracellular synthesis of angiotensin II and conversion of angiotensin I. Since the angiotensin-II effect could be prevented by intracellular administration of the receptor antagonist DuP 753, De Mello [1994] concluded that there is an intracellular angiotensinII receptor involved in the regulation of gj. Taken together these investigations point to a possible influence of the renin-angiotensin system on cardiac cellular coupling. According to De Mello [1992, 1994] it can be argued that at least in parts the positive effects of

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angiotensin-converting enzyme inhibitors and protective effects in regional myocardial ischemia can be attributed to this improvement in intercellular communication. In the light of the theory of arrhythmia formation focussing on the generation of reentry by slowing conduction and by enhanced dispersion, this coupling effect of an angiotensin-converting enzyme inhibitor can be seen as an antiarrhythmic action. In 11-month-old cardiomyopathic hamsters the uncoupling action of angiotensin II was most pronounced in poorly coupled cells and could be enhanced by enalaprilat [De Mello, 1996]. The author concluded that the decrease in intercellular coupling in cardiomyopathy may in part be due to an activation of the cardiac renin-angiotensin system. Renin, angiotensin I, angiotensin II and angiotensin-converting enzyme have been found in cardiomyocytes using immunofluorescent staining [Dostal et al., 1992] with the enzyme being located in the perinuclear region. Another group of drugs affecting gap junctional conductance are the antiarrhythmic peptides [for a detailed review see, Dhein and Tudyka, 1995]. In 1980 a hexapeptide with a molecular weight of 470 was isolated from bovine atria by Aonuma et al. [1980b]. This peptide improved synchronization of embryonic chick heart cell aggregates, and was thus proposed to possess antiarrhythmic actvity. From bovine atria 200 lg/kg wet tissue of the pure peptide were yielded. Its antiarrhythmic action was established in neonatal rat cardiomyocytes. Fibrillation induced by either ouabain, 3 mmol/l Ca2+ or 0.7 mmol/l K+ was converted to regular beating by 0.1 mg/l of the peptide. If added to the cell culture medium it increased the number of beating centers, the relative content of spreading cells and protein synthesis [Aonuma et al., 1980a]. In later studies the antiarrhythmic peptide (10 mg/kg) was shown to be effective in vivo against CaCl2-induced and aconitine-induced arrhythmia in mice [Kohama et al., 1987]. Moreover it prevented fibrillation in dogs and rats [Aonuma et al., 1983]. In an ouabain and an ADP model the time to the onset of arrhythmia was prolonged by the antiarrhythmic peptide, while it failed to prevent epinephrine-induced arrhythmia. In subsequent investigations the amino acid sequence was determined as H2N-Gly-Pro-4Hyp-Gly-Ala-GlyCOOH [Aonuma et al., 1982] and tissue levels could be measured using a radioimmunoassay in the heart (203 pmol/g), kidney (165 pmol/g) and blood (3.8 pmol/g) [Kohama et al., 1985]. Interestingly, in the course of CaCl2- and aconitine-induced arrhythmias the tissue levels in the heart, but not the kidney, increased, whereas in epinephrine-induced arrhythmia the tissue level was found to have decreased [Kohama et al., 1986]. In contrast, plasma levels were increased 3-fold in all 3 forms of arrhythmia. Until that point the mechanism of action of the peptide remained unclear. The first investigation directed toward the elucidation of the underlying mechanism of action revealed that the antiarrhythmic peptide did not alter depolar-

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ization velocity, action potential amplitude, duration or shape and did not exhibit any action on muscarinic receptors in canine Purkinje fibers [Argentieri et al., 1989], so that the authors concluded that the mechanism consisted of effects on passive membrane properties or other actions rather than an action on membrane ionic currents. From the experiments carried out by Dhein et al. [1994, 1995b, 1996] and Mu¨ller et al. [1997a, b] it was concluded that the antiarrhythmic peptide and a synthetic derivative improve cellular coupling by an increase in gap junctional conductance. Dhein et al. [1994] assessed the effects of the antiarrythmic peptide and several synthetic derivatives, synthesized according to the Merrifield technique using the Fmoc strategy, in isolated rabbit hearts submitted to regional ischemia. The action of the antiarrhyhtmic peptides under normal conditions was a reduction in the dispersion of the action potential duration measured at 256 ventricular unipolar electrodes. The antiarrhythmic peptide AAP10 (H2N-GlyAla-Gly-4Hyp-Pro-Tyr-CONH2), an amide, was found to be the most effective with an onset of action at 0.1 nmol/l and maximum effect at 10 nmol/l. The action consisted of a homogenization of the action potential duration so that local differences became smoothed without affecting the mean action potential duration as shown in figure 22. The peptide did not exhibit any other influence on cardiac parameters (e.g. left ventricular pressure, coronary flow, QRS duration, PQ time). If hearts were submitted to regional ischemia by LAD occlusion for 30 min, pretreatment with 10 nmol/l AAP10 led to a significant reduction in the ischemia-induced alterations in the activation patterns and to a reduction in the incidence of ventricular fibrillation, especially of late phase VF (type Ib) [Dhein et al., 1994, 1995b, 1996]. In order to clarify the mechanism of action Dhein et al. [1994] investigated the effects of AAP10 on the transmembrane action potential in isolated papillary muscles of guinea-pig heart. They found no effect on action potential duration and morphology, on action potential amplitude, on maximum upstroke velocity or on resting membrane potential in concentrations up to 1 lmol/l. However, they observed a reduction in the coupling time within 1 min after application, i.e. in the interval between the stimulus and the propagated action potential. The effect was reversible on wash out. Because due to the lack of effect on the maximum upstroke velocity an effect on the sodium current could be ruled out, the reduction in coupling time was a first strong hint of a possible action on the gap junctions. Thus, the authors decided to investigate the effect of the antiarrhythmic peptide AAP10 on gap junctional current in adult guinea-pig ventricular cardiomyocytes directly using the double-cell voltage clamp (whole cell patch configuration). In these experiments they found an improvement in gj by 10 nmol/l AAP10. It took about 2 minutes until the onset of this effect. The effect could be

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Fig. 22. Reduction in dispersion of the ventricular action potential duration by the synthetic antiarrhythmic peptide AAP10. The distribution of the action potential duration (assessed as the epicardial activation-recovery interval, ARI) on the surface of an isolated rabbit heart before and after treatment with AAP10. Note the greater variability of the epicardial action potential duration (ARI) before administration of AAP10 [Dhein et al., 1997c].

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Fig. 23. Survey of the various pharmacological interventions at the gap junctional coupling. For details see text.

washed out within several minutes [Dhein et al., 1995b; Mu¨ller et al., 1997a, b]. They showed that the spontaneous decline in gj by Ö2.5 nS/min was reversed by AAP10, so that an increase of 1 nS/min was seen. In subsequent experiments guinea-pig papillary muscles were submitted to hypoxia and glucose-free perfusion, so that they uncoupled after 12 min. This could be prevented by pretreatment with 10 nmol/l AAP10 [Dhein et al., 1997b; Mu¨ller et al., 1997b]. Since in such low concentrations only a very slight effect on coupling time was seen under normoxic conditions, the authors concluded that AAP10 might preferentially act on uncoupled cells. With regard to the antiarrhythmic action they favored the hypothesis that improvement in gap junctional coupling reduces action potential dispersion and prevents slowing of conduction by uncoupling, thereby preventing arrhythmia. This is in line with a stabilization

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of the activation patterns under ischemic conditions as observed [Dhein et al., 1994, 1996, 1997c]. What are the therapeutic implications? According to De Mello [1986b], cellular coupling is mainly influenced by the intercellular axial resistivity in the direction of propagation (Ri) and is inversely dependent on the nonjunctional sarcoplasmic membrane resistance (Rm). As was described in the foregoing chapters Ri can be altered by a large number of factors and diseases. Changes in cellular coupling can be expected to alter conduction velocity and synchronization of the cells. Because under normal conditions the cells are more or less well coupled, it can be assumed that in most pathological states (e.g. regional ischemia, heart failure, acidosis, hypoxia, Chagas disease and others) coupling will be reduced resulting in slowing of conduction and possible enhancement of dispersion, which both can make the heart more prone to reentrant arrhythmia. Thus, it can be imagined that such drugs, enhancing cellular coupling, may prevent arrhythmia in these states characterized by reduced coupling. However, until now they have only been shown to exert a prophylactic effect and it is questionable whether enhancement of coupling during manifest arrhythmia is more effective than the existing classic antiarrhythmics. In situations with uncoupling being due to structural changes such as fibrosis, drugs which can improve gap junctional coupling are probably ineffective since they can enhance coupling only in functional gap junctions. In these situations one can speculate whether enhanced expression of gap junctions might be useful but it has not yet been shown experimentally. The antiarrhythmic peptides have been shown to be effective in the prophylaxis of ischemia-associated ventricular fibrillation (type-Ib arrhythmia) [Dhein et al., 1994, 1996] and ouabain-induced arrhythmia [Aonuma et al., 1980b], a state which is known to be associated with uncoupling [De Mello, 1976]. According to the considerations at the beginning of this chapter, it might be advantageous that at least AAP10 seems to act preferentially in uncoupled cells. For the problem of prophylactic antiarrhythmic treatment these antiarrhythmic peptides are probably not the final solution. Because of their peptide nature they are not well suited for in vivo studies and they are probably only indicated for the prevention of arrhythmias due to reduced coupling, but they are a first step in the direction of a new class of drugs influencing gap junctional coupling. The various pharmacological approaches to the modulation of gap junctional coupling are summarized in figure 23.

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8

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Methods for Investigation of Gap Junctions

In this chapter more detailed information on the double-cell voltageclamp setup and protocols for assessing gap junctional conductivity is given, as well as a description of the cell-isolation procedure for this purpose and cell culture models. Information on immunocytochemical localization of gap junctions and on the experimental procedure of preparing specimens and slides for immunohistology is given. A protocol for isolation of gap junction proteins is also outlined. Readers interested in more details of the cell-culture technique regarding incubators, sterile technique, etc., and different isolation and culture protocols are referred to more specialized literature [Lindl and Bauer, 1994; Piper, 1990]. Many experiments on gap junctions have been carried out using the neonatal rat heart cells. Thus, the procedure of isolating and culturing these cells (as used in the author’s laboratory) will be discussed below.

8.1 Culture of Neonatal Rat Cardiomyocytes Prepare the following solutions: Coating solution M199 (with Earl’s salts) 100 lg/ml penicillin 100 lg/ml streptomycin 10% fetal calf serum (FCS) PBS/glucose solution NaCl 137 mmol/l KCl 2.7 mmol/l 8.3 mmol/l Na2HPO4 KH2PO4 1.5 mmol/l Glucose 20 mmol/l pH to be adjusted to 7.4 Desaggregation solution Phosphate-buffered saline (PBS) Glucose Bovine serum albumin (BSA) Collagenase type II (Gibco)

50 200 500 50

ml mg mg mg (204 U/mg)

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Medium for resuspension of supernatants M199 (with Hanks’ salts and HEPES) 100 lg/ml penicillin 100 lg/ml streptomycin 10% FCS 25 mmol/l HEPES 2 mmol/l L-glutamine Culture medium for the 1st day M199 (with Earl’s salts and HEPES) 2 mmol/l L-glutamine 5% FCS 100 lg/ml penicillin 100 lg/ml streptomycin Culture medium after the 1st day M199 (with Earl’s salts and HEPES) 2 mmol/l L-glutamine 1% FCS 100 lg/ml penicillin 100 lg/ml streptomycin Dulbecco’s wash solution for the 2nd day NaCl 137 mmol/l KCl 2.68 mmol/l 6.48 mmol/l Na2HPO4·2H2O 1.47 mmol/l KH2PO4 0.49 mmol/l MgCl2·6H2O 0.81 mmol/l MgSO4·7H2O 0.9 mmol/l CaCl2 pH to be adjusted to 7.2 The culture dishes have to be prepared 24 h before use. They have to be coated with the coating medium. Neonatal rats (1–2 days old) are killed by decapitation and then sprayed with 70% ethanol for desinfection. After thoracotomy the pericard is opened and the heart removed. Bath the organ in ice-cold PBS/glucose solution in a Petri dish to remove blood. Remove atria, transfer the heart to another Petri dish, chop up the ventricles with two sterile scalpels and incubate in 7 ml desaggregation solution and stir gently (140 rpm) at 37 ºC. Allow sedimentation of the tissue and remove the supernatant. Add fresh dissociation solution and repeat this procedure 6 times. Suspend the supernatants in the medium for resuspension of supernatants (each in 8–9 ml, ice-cold). Centrifuge these cell solutions for 5 min at 700 rpm and resuspend the pellet in culture medium for the 1st day. Seed the cells in 25-cm2 plastic flasks and incubate at 37 ºC/5% CO2. After 2 h of preplating (this time is required for nonmuscular cells to attach) the supernatant of this flask is filtered through a nylon mesh (pore width 100 lm) and then seeded in Petri dishes at a density of about 10,000 to 100,000 cells/cm2 in culture medium for the 1st day. After 24 h the Petri dishes are rinsed off with Dulbecco’s wash solution and culture medium for the 1st

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day (5% FCS) is added. 72 h after preparation half of this medium is removed and replaced by the 1% FCS medium. Thereafter (i.e. in the following days), cells are incubated with the 1% FCS medium only. One may add 10% horse serum in order to inhibit fibroblast growth.

8.2 Culture of Embryonic Chick Cardiomyocytes Cultured embryonic chick heart cells often form coupled cell pairs or aggregates which may be studied using the double-cell voltage-clamp technique. In the following an isolation and culture protocol is given as used in the author’s laboratory. Besides this, other protocols may also suit. Prepare the following solutions: Glutamine solution Glutamine 200 mmol/l Hanks’ buffered saline solution (HBSS ) NaCl 8,000 mg/l KCl 400 mg/l 60 mg/l KH2PO4 47.5 mg/l Na2HPO4 350 mg/l NaHCO3 Glucose 1,000 mg/l pH 7.4 Trypsin solution (0.25%) Trypsin 2.5% in HBSS (+phenol red) HBSS Culture medium FCS Horse serum Glutamine solution Penicillin Streptomycin M199

3 ml 27 ml

4% 2% 3.4 ml/l 100 IU/ml 100 IU/ml add 1 liter

All material used for isolation and culture of the cells must be sterile, all media have to be autoclaved. Take 10 eggs (5–7 days old; make sure that they are kept at the same temperature until the start of the procedure), wash with 70% ethanol and open at the egg’s pole under sterile conditions in laminar flow. Decapitate the embryo, isolate the heart from the embryo and remove the atria. Bath the ventricles in 5–10 ml HBSS at room temperature in a Petri dish. Chop up the ventricle with sharp scalpels and transfer to a watch-glass. Add some milliliters of the 0.25% trypsin solution at 37 ºC for 7 min. Thereafter mix the solution well and filter through gauze with a mesh size of 100 lm in a Falcon tube containing 10 ml HBSS (4 ºC) and 4% FCS. Centrifuge for 5 min at 1,500 rpm and 4 ºC. Resuspend the pellet with HBSS, centrifuge again and repeat the procedure once again. Dissolve the pellet in about 10 ml

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culture solution (M199 with supplement as described) so that a cell density of nearly 500,000 cells/ml is yielded. Seed in plastic cell culture flasks and incubate for at least 24 h. After this time, aggregates can be observed which exhibit spontaneous activity and in the periphery of these aggregates cell pairs are often found. The medium has to be changed every 24 h.

8.3 Isolation of Adult Guinea-Pig Cardiomyocytes If adult cardiomyocyctes are used for the double-cell voltage-clamp technique, it is desirable to yield a high amount of cell pairs. Therefore, protocols using proteases such as trypsin should not be used, because proteases may enhance separation of the cells and disrupt or destroy the gap junctions. Similarly, protocols using proteases are critical if ion channels or receptors with large extracellular protein domains are to be investigated in freshly isolated cells. In the author’s laboratory a collagenase protocol is commonly used to isolate adult guinea-pig cardiomyocyte pairs, and is described below. It should be noted that this is surely not the ‘only true’ protocol, but it is a suitable one. First of all the following solutions have to be prepared. Solution A 3 mol/l NaCl 3 mol/l KCl 0.1 mol/l KH2PO4 0.1 mol/l MgSO4 0.4 mol/l NaHCO3 0.5 mol/l HEPES/Na Glucose H2O

21 ml 783 ll 6 ml 12.5 ml 31.1 ml 5 ml 0.54 g add 500 ml

Solution B Pyrovate 44 mg BSA 200 mg Solution A add 200 ml Equilibrate with 95% O2 and 5% CO2, adjust pH to 7.4 Solution C 1 vial collagenase Worthington type 100 U/ml 12.5 ll 0.1 mol/l CaCl2 Solution B add 10 ml Solution D Solution B 20 ml 0.1 mol/l CaCl2 10 ml Equilibrate with 95% O2 and 5% CO2 Solution E (only for short-term culture of the cells) FCS 10% 1 ml Penicillin 2.4 mg (or 100 IU/ml) Streptomycin 4 mg (100 IU/ml) Glutamine 200 mmol/l 100 ll M199 add 20 ml

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After the guinea pig (250–350 g) has been killed, the heart is prepared according to the Langendorff technique and transferred to a Langendorff apparatus and perfused at 37 ºC for 10 min with solution B (i.e. Ca2+-free saline). Thereafter, the heart is perfused with solution C in a recirculating manner for about 30 min. It is important to make sure that both solutions do not get mixed. While perfusing with the collagenase solution, the heart becomes ‘slimy’ and a little transparent. During this phase the perfusion rate often has to be reduced. At the end of this period the heart is removed from the apparatus, atria are removed and the remaining heart is cut into small pieces using two scalpels. The tissue is transferred to a glass test tube and dissolved with the rest of the collagenase solution, slightly gassed with 95% O2 and 5% CO2 at 37 ºC. Next, the solution containing the tissue is filtered through gauze with a mesh size of 200 lm and centrifuged for 1 min at about 600 rpm. After removing the supernatant, the pellet is resuspended in 10 ml solution D (37 ºC; no longer gas the solution). Again the solution is centrifuged for 1 min at 600 rpm and the pellet resuspended in solution D. After 1 min the calcium concentration in the solution is gradually increased (this is a critical phase for the cells, because during this procedure many cells are impaired) by adding 5 ll 0.1 mol/l CaCl2, 7.5 ll after another minute, 7.5 ll after the next minute and 10 and 15 ll in the following 2 min, thereby adjusting the calcium concentration finally to about 0.45 mmol/l. Following these steps, the solution is centrifuged a last time for 1 min at 600 rpm and the resulting pellet is either resuspended in 20 ml solution B (with additional Ca2+) or in Tyrode solution, if the cells are to be used immediately for an experiment, or the pellet is dissolved in solution E for cultivating the cells. For this purpose 2 ml of the cell-containing solution are dissolved with 5 ml solution E in Petri dishes (3 cm diameter) and kept in the incubator for a maximum of 3 days. However, it should be noted that the content of cell pairs gradually declines with time and is maximum shortly after isolation.

8.4 Immunohistochemistry To detect gap junction proteins and their distribution within the tissue, immunohistochemical methods are commonly used. The best results are obtained with frozen sections, since other methods such as paraffin embedding or acrylate embedding may affect the antigeneity of the proteins to be detected (this is because tissue fixation, for example, glutaraldehyde or formaldehyde cross-link proteins), although several laboratories have also used these embedding techniques with success. If tissue has to be transported prior to freezing, for example from the clincal theater to the laboratory, it is recommended to keep the delay to freezing as short as possible and to transport the tissue in cooled (5 ºC) tissue culture medium (such as RPMI) or in cooled saline. To freeze the tissue the following protocol can be used. Perfuse the heart with a mixture of saline-buffered solution (e.g. Tyrode’s solution) and glycerine (1:1) Let the fluid drain Transfer the heart to a methanol bath (pre-cooled to Ö70 ºC) Let the methanol drain Transfer the tissue to fluid N2

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In the next step sections have to be prepared from the tissue block. Frozen sections should be cut at 5-lm thickness and be picked on aminopropyltriethoxysilane (APES)-coated slides (alternative: Superfrost slides) using a cryostat at Ö25 ºC. If small tissue samples are used, these can be embedded in Tissue-Tek to facilitate sectioning. The sections have to be air-dryed overnight. Thereafter, sections are fixed in acetone for 20 min (alternatively in methanol, 30 min). Thereafter, sections are incubated for 30 min in Triton X–100 (0.1%; this step is optionally, but can be used to permeabilize the tissue or cultured cells if the antibody binds to intracellular-binding sites, and can facilitate access of the antibody to the binding site). This is followed by coating with 1% BSA (fatty acid free) in PBS at pH 7.5 for 20 min for blocking unspecific protein-binding sites in the specimen. Next, the section is exposed to the primary antibody (e.g. monoclonal mouse anti-rat Cx43, epitope: amino acids 252–270; Biermann GmbH, Bad Nauheim, Germany) at a working dilution of 1:100 for 1 h. Thereafter, the specimen has to be washed in order to remove antibody which was not bound by specific sites (wash with PBS and 1% BSA for 20 min, change wash solution 3 times). In the next step the secondary antibody has to be coupled to the primary in order to make it visible. In our laboratory, we use FITCcoupled secondary antibodies or DTAF-coupled (DTAF has the advantage of less bleeching during exposure to the excitation light; for the primary antibody mentioned above a FITClabeled goat anti-mouse IgG antibody, Sigma was used in the author’s laboratory. Using fluorescence microscopy is a very elegant procedure which should be preferred if possible. However, in some cases problems may arise from unspecific fluorescence or, for certain investigations, from the fact that the structure of the tissue cannot be visualized simultaneously. In such cases, alternatively, secondary antibodies coupled to peroxidase or to alkaline phosphatase can be used. However, these have the disadvantage that unspecific reactions with tissue components are possible. A possible alternative is the use of the streptavidin-biotin method (see below). Next, the specimens are incubated with the secondary antibody for another hour at working dilutions of 1:300 to 1:1,000. Thereafter, the slides are washed with PBS at pH 7.5 for 20 min, embedded in karyon and covered with a coverslip, dryed at 4 ºC (overnight, keep in the dark) and sealed with acryl varnish (nail varnish is also suitable). FITC and DTAF are both excited with blue light (at k>470 nm; excitation filters: 2·SP 490+2 mm LP 455) and emit green fluorescent light (at k>540 nm; emission filter; barrier filter; LP 515). Protocol for immunostaining (as used in the author’s laboratory) 30 min 0.1% Triton X-100 20 min 1% BSA in PBS (pH 7.5) 1 h primary antibody (1:100) 20 min PBS+1% BSA (pH 7.5) 1 h secondary antibody 1:300 to 1:1,000 20 min PBS (pH 7.5) Embed in karyon, cover with coverslip, dry at 4 ºC and seal with acryl varnish PBS: 8 g NaCl, 0.2 g KCl, 1.15 g Na2HPO4, 0.2 g KH2PO4, add H2O to a final volume of 1 liter. Adjust pH as desired. The antibody stock solution should contain 1% BSA in PBS. In the following, protocols for alkaline phosphatase-coupled, peroxidase-coupled antibodies and for the streptavidin-biotin system are given [Jackson and Blythe, 1993; Ormerod and Imrie, 1989].

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If a biotinylated secondary antibody is used for the streptavidin-biotin method the following protocol is suitable for frozen sections: Air-dryed frozen sections have to be rehydrated in Tris-buffered saline (TBS) Let the fluid drain Incubate with primary antibody in TBS for 1 h Wash with TBS (20 min, 3 times) Let the fluid drain Incubate with the biotinylated secondary antibody (30 min, antibody dilutions of 1:200 are often suitable) Wash with TBS (20 min, 3 times) Make streptavidin-biotin/horseradish peroxidase complex from: 20 ll streptavidin, 20 ll biotinylated horseradish peroxidase, 1 ml TBS (shake well and wait 30 min) Let the fluid drain from the sections and incubate with the streptavidin-biotin/horseradish peroxidase complex for 30–60 min Wash with TBS (20 min, 3 times) Develop the peroxidase in 3,3-diaminobenzidine tetrahydrochloride (DAB) solution: 3 ml DAB stock solution and 12 drops hydrogen peroxide (30% w/v) are dissolved in 400 ml TBS; incubate the specimen with this solution for 10 min (perhaps longer, control with microscope) Wash with tap water Incubate with copper sulfate solution for 5 min Wash with tap water Counterstain as desired Embed in karyon, cover with coverslip, dry and seal with acryl varnish TBS (0.5 mol/l, pH 7.6): 60.5 g tris(hydroxyl)methylamine are dissolved in 750 ml H2O, pH is adjusted to 7.6 with HCl, 85 g NaCl is added with H2O to a final volume of 10 liters DAB stock solution: 7.5 g DAB in 300 ml Tris buffer at pH 7.6 (caution, DAB is carcinogenous) Copper sulfate solution: 4 g CuSO4, 7.2 g NaCl, add to 1 liter H2O For peroxidase-coupled secondary antibody the following protocol may be used: Air-dryed frozen sections have to be rehydrated in TBS Let the fluid drain Incubate with primary antibody in TBS for 1 h Wash with TBS (20 min, 3 times) Let the fluid drain Block the endogenous enzyme with 0.3% H2O2 for 30 min (alternatively, incubate in 2.3% periodic acid for 5 min, wash with water, rinse in 0.03% potassium borohydride and wash), or incubate for 5 min in 0.1% phenylhydrazine in PBS) Incubate with the peroxidase-conjugated secondary antibody (30 min) Wash with TBS (20 min, 3 times) Develop the peroxidase in DAB solution: 3 ml DAB stock solution and 12 drops hydrogen peroxide (30% w/v) are dissolved in 400 ml TBS, incubate the specimen with that solution for 10 min (perhaps longer, control with microscope) Wash with tap water Incubate with copper sulfate solution for 5 min Wash with tap water

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Counterstain as desired Embed in karyon, cover with coverslip, dry and seal with acryl varnish Alternatively alkaline phosphatase-coupled secondary antibody can be used with an antialkaline phosphatase antibody (APAAP technique). For this method the following protocol may be used: Air-dryed frozen sections have to be rehydrated in TBS Block the endogenous enzyme with 20% acetic acid for 5 min Wash well with water Let the fluid drain Incubate with primary antibody in TBS for 1 h Wash with TBS (20 min, 3 times) Let the fluid drain Incubate with the alkaline phosphatase-conjugated secondary antibody (30 min) Wash with TBS (20 min, 3 times) Wash with water Develop the alkaline phosphatase with fast red: dissolve 5 mg sodium naphthol AS BI phosphate in dimethylformamide (few drops) and add to 5 mg fast red TR salt in 10 ml veronal acetate buffer (pH>9.2), incubate the slides for 1 h Wash with tap water Counterstain as desired Embed in karyon, cover with coverslip, dry and seal with acryl varnish It should be noted that there is not the ‘one’ protocol for immunohistology, but many variations and the investigator has to find the most suitable for his purposes by varying the several steps. However, some problems often encountered with immunohistology will be discussed briefly. The antibodies may bind to unspecific protein-binding sites which can be avoided with blocking agents such as BSA and gelatin. Some antibodies may react with charged surfaces or charged proteins. This can be reduced with surfactant reagents as Tween 20 and NaCl. In the tissue there can be Fc fragment-binding sites which can be blocked by incubation with serum. Alternatively the F(ab)2 fraction of antibody may be used. It may be possible that the antibody used can react with the same or a similar epitope in other proteins of the host tissue (especially with interstitial areas). Cross-reactivity from the diluted second antibody may be absorbed by adding to it 1–2% of serum of the host species. Antibodies and streptavidin may be bound by basic amino acids of the tissue. This can be inhibited by adding 2 mg/ml of the basic peptide poly-L-lysine (MW 3,000–6,000) to the diluted antibody. Disturbing autofluorescence may be reduced by staining the tissue with pontamine sky blue (0.05% in PBS with 1% dimethylsulfoxide) for 30 min before applying the first antibody. Controls have to be carried out: incubation (and development without incubation with the primary antibody, i.e. only with secondary antibody) to control for nonspecific reactions of the second antibody. In addition, if immunofluorescence is used, unstained slides have to be investigated also as controls in order to detect nonspecific background fluorescence (for example of connective tissue), which may in some cases be enhanced by some drugs which were administered prior to taking the sample. Using immunohistology, it is possible to determine the distribution of a specific connexin within the tissue or within cells. It can also be used for semiquantitative evaluations by

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counting, for example, the percentage of positively stained cells among all cells in a view field, or measuring the positively stained membrane length in comparison with the total membrane length. This has to be repeated with many sections and many view fields, and will then give reliable results. A quantification of the protein content by measuring the intensity of staining (e.g. of immunoflourescence) is only possible if the primary antibody is labeled, as it cannot be assessed how many molecules of the labeled secondary antibody will bind to the primary. However, such quantitative immunofluorescence is extremely error prone and difficult, and great caution has to be taken in order to garantee that there is no difference in bleeching between the samples, the optical system is unchanged, the background is corrected exactly in the same way and so on. Thus, if quantification of the connexin content is desired, other methods are recommended, as the direct isolation of connexin (see next section).

8.5 Isolation of Cx43 Often the question arises, whether the total amount of connexin is altered. Because immunohistology is mostly a semiquantitative method, the following biochemical method for isolation of gap junctions may be a suitable alternative. After isolation of the gap junction pellet SDS-PAGE has to be carried out and the gels have to be stained. For control an immunoblot is recommended. Protocol for isolation of gap junctions (as used in the author’s laboratory, adapted from Manjunath et al. [1982]). The quantities refer to the initial tissue amount of a rabbit heart. All steps have to be carried at 0–4 ºC if not stated otherwise. 1 Transfer tissue (heart) to cold 1 mmol/l NaHCO3 (pH 8.2) 2 Remove superficial fat and vessels 3 Cut the tissue into small pieces and add these to 50 ml 1 mmol/l NaHCO3 (pH 8.2) 4 Add phenylmethylsulfonyl fluoride (PMSF) to a final PMSF concentration of 1 mmol/l 5 Stir 15 min at 4 ºC 6 Homogenize tissue: 60 s, Vmax (Virtis homogenizer) 7 Further homogenization using tissue mizer, SDT 100 EN 8 Dilute the homogenate to 300 ml with 1 mmol/l NaHCO3 (pH 8.2), and filter through 6–8 sheets of medical gauze 9 Centrifuge the filtrate: 15 min, 33,000 g 10 Remove the supernatant and disolve the resulting pellet in 300 ml 1 mmol/l NaHCO3 (pH 8.2) 11 Centrifuge: 15 min, 33,000 g 12 Remove supernatant and resuspend pellet in 100 ml 0.6 mol/l KI, 6 mmol/l Na2S2O3 in 1 mmol/l NaHCO3 (pH 8.2) 13 Add PMSF (as step 4) 14 Stir overnight at 4 ºC 15 Filter through 6 sheets medical gauze 16 Centrifuge: 30 min, 27,000 g 17 Remove supernatant, resuspend the pellet in 100 ml 0.6 mol/l KI, 6 mmol/l Na2S2O3 in 1 mmol/l NaHCO3 (pH 8.2), homogenize in homogenizer (3 strokes only)

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18 Centrifuge: 30 min, 27,000 g 19 Remove supernatant, wash pellet in 70 ml (final volume) 0.6 mol/l KI, 6 mmol/l Na2S2O3 in 1 mmol/l NaHCO3 (pH 8.2) 20 Centrifuge: 15 min, 27,000 g 21 Remove supernatant, wash pellet in 30 ml 5 mmol/l Tris (pH 10) at room temperature 22 Homogenize the solution in homogenizer (3 strokes only) 23 While gently stirring add 30 ml 0.6% N-lauroylsarcosine in 5 mmol/l Tris (pH 10) 24 Stir at room temperature for 10 min 25 Prepare a density gradient in a centrifuge tube and add the solution, from top to bottom: 20 ml sample volume; 8 ml 35% sucrose, 0.3% deoxycholate in 5 mmol/l Tris (pH 10), and 5 ml 44.5% sucrose, 0.3% deoxycholate in 5 mmol/l Tris (pH 10) 26 Allow to stand for 20 min at room temperature 27 Centrifuge: 60 min, 65,000 g, 15 ºC 28 Take the band at the 35–44.5% interface and dissolve in 45 ml 0.3% deoxycholate in 5 mmol/l Tris (pH 10) 29 Prepare a density gradient and add the solution, from top to bottom: 15 ml sample volume; 9 ml 35% sucrose, 0.3% deoxycholate in 5 mmol/l Tris (pH 10), and 9 ml 44.5% sucrose, 0.3% deoxycholate in 5 mmol/l Tris (pH 10) 30 Centrifuge: 60 min, 65,000 g, 15 ºC 31 Take the band at the 35–44.5% interface and dissolve in 45 ml 0.3% deoxycholate in 5 mmol/l Tris (pH 10) 32 Repeat step 29 with 15 ml of the solution 33 Take the band at the 35–44.5% interface and dissolve 1:1 in 0.3% deoxycholate in 5 mmol/l Tris (pH 10) 34 Centrifuge: 30 min, 106,000 g, 15 ºC 35 Wash the pellet in 5 mmol/l Tris (pH 10) 36 The pellet contains the gap junctional pellet contaminated with nonjunctional membranes 37 Prepare a density gradient and add the solution, from top to bottom: sample; 9 ml 31.5% sucrose, 0.3% deoxycholate in 5 mmol/l Tris (pH 10), and 9 ml 35% sucrose, 0.3% deoxycholate in 5 mmol/l Tris (pH 10) 38 Centrifuge: 90 min, 65,000 g, 15 ºC 39 Take the material at the 31.5–35% interface 40 Centrifuge: 30 min, 106,000 g, 15 ºC 41 Wash as step 35 42 Pellet: gap junction pellet Following this isolation of the gap junction pellet the connexin has to be isolated using discontinuous gel electrophoresis (SDS-PAGE). The stacking gel and separating gel can be prepared according to the following recipies (as used in the author’s laboratory for Maxigel, Biometra, Germany): Stacking gel 5% (volume 6 ml) Acrylamide: 30% (w/v), 0.8% (w/v) bisacrylamide in water: 1 ml Tris/HCl: 0.625 mol/l (pH 6.8): 1.2 ml Sodium dodecyl sulfate (SDS): 0.5% (w/v) in water: 1.2 ml H2O: 2.6 ml

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Ammonium persulfate: 10% (w/v) in water: 30 ll N,N,N,N-tetramethylethylenediamine (TEMED): 6 ll Separating gel 12.5% (volume 30 ml) Acrylamide: 30% (w/v), 0.8% (w/v) bisacrylamide in water: 12.5 ml Tris/HCl: 1.88 mol/l (pH 8.8): 6 ml SDS: 0.5% (w/v) in water: 6 ml H2O: 5.5 ml Ammonium persulfate: 10% (w/v) in water: 150 ll TEMED: 30 ll Sample buffer 0.5 mol/l Tris (pH 6.8) 1.2 ml 1 ml 20% SDS in H2O Glycerin 1 ml 5% b-Mercaptoethanol 0.1% (v/v) bromphenol blue in ethanol 0.5 ml The sample buffer is distributed in Eppendorf tubes in amounts of 500 ll. Finally, 25 ll bmercaptoethanol is added prior to use. Running buffer 0.18 mol/l Tris (12.1 g/l) 0.1 mol/l glycin (7.5 g/l) 3 mmol/l SDS (1 g/l) Staining solution 500 ml methanol 500 ml concentrated acetic acid 100 ml H2O 2.5 g Coomassie brillant blue R-250 Destaining wash solution 50 ml methanol 75 ml concentrated acetic acid Prepare a 1-mm gel from these gel solutions with 10 indentations for 50-ll samples. Fill the indentations with running buffer and add 40 ll of sample (the sample is dissolved 1:1 in sample buffer). It is necessary to reserve one lane for a molecular weight marker. To run the stacking gel 25 mA is used, for the running gel 60 mA (this refers to Maxigel, Biometra, Germany). After running, the gel is bathed in the staining solution for 30 min. Thereafter, it is washed in the destaining wash solution for 24 h. Finally the gel is dryed at 60 ºC for 90 min.

8.6 Double-Cell Voltage-Clamp Very often it is necessary to measure the conductance of gap junctional channels. In principle, there are two possibilities: one can measure the single-channel conductance, or the total conductance between two cells. It is important to make this choice before setting

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up the experiment, because for these two kinds of investigation, different amplifiers may be used. As pointed out in a previous chapter the serial resistance of the pipettes may disturb accurate measurement of the total resistance between two cells. The problem arises from the change in potential before and after the pipette (which serves as a resistor) during current injection via this serial resistance. According to a paper by Wilders and Jongsma [1992] the series resistance resulting from the pipette and from the cytoplasm can make it difficult or even impossible to accurately measure the voltage dependence of gap junctions. This series resistance problem can be avoided by the use of discontinuous singleelectrode voltage-clamp (dSEVC) amplifiers or switch-clamp amplifiers. These amplifiers switch between voltage measurement and current injection thereby avoiding artefacts by the resistance of the pipettes. The use of switch-clamp amplifiers is, from a present point of view, recommended for the measurement of the total conductance between two cells. However, it should be noted that the use of these amplifiers is a bit more complicated than that of normal patch-clamp or voltage-clamp amplifiers, since the switching frequency, gain, duty cycle and capacity compensation have to be adjusted very accurately. Since voltage should be measured after the injection of current, it is necessary to adjust the system in a way that the voltage developed on the microelectrode resistance and capacitance by the injected current is decayed prior to voltage sampling. With accurate capacity compensation it is possible to measure the membrane potential at a time when no current passes the recording electrode. However, switch-clamp amplifiers are often problematic if single-channel conductance is to be measured. Some can be used for this purpose with specially designed head stages. Most investigators use classic patch-clamp amplifiers to measure single-channel events. In the author’s laboratory gap junction conductance between two cells is measured using the double-cell voltage-clamp method with two switch-clamp amplifiers (SEC-05, NPI Electronic, Germany) in a double whole-cell patch. The procedure is described below. The double-cell voltage-clamp setup (as used in the author’s laboratory) consists of the following components: 2 SEVC amplifiers 1 inverted microscope (objectives 10¶, 20¶, 40¶, 100¶, all long-working distance with correction for thickness of the glass ground of the bath; eyepiece: 10¶; phase contrast) 2 micromanipulators with electronic remote command 1 break-out box (for connection of the amplifiers to the computer) 1 PC system equiped with an A/D converter Software for controlling the SEVC amplifiers (make sure that your software can control two amplifiers; it is preferable if the software can record 2 voltage and 2 current traces) Bath with inlet and outlet (it is recommended to use a bath with temperature control) Roller pump Tubings Ground Faraday cage Pipette puller Microforge It is absolutely necessary to provide proper grounding of all components in the setup. The microscope, the manipulators and the bath have to be shielded by a Faraday cage. The amplifiers, the PC system, oscilloscopes and roller pump are located outside the cage. All components of the setup have to be connected to a star point. This is often represented by

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a solid copper bar with banana jacks. No component should be coupled (directly or indirectly) more than once to the star point. Otherwise, so-called grounding loops will be created which cause noise. The star point itself is connected to the ground of the building. Problems often arise from saline-filled tubings. Ingoing and outgoing tubings should be interrupted by some sort of a dropper as used with common infusion tubings. Isolate or cultivate cells according to the protocols given before. Pull pipettes of 2–3 MX resistance, fire-polish in a microforge or treat with sylgard. Prepare the following solutions. Extracellular solution NaCl 135 mmol/l KCl 4 mmol/l 2 mmol/l CaCl2 1 mmol/l MgCl2 0.33 mmol/l NaH2PO4 HEPES 10 mmol/l Glucose 10 mmol/l pH 7.4 Pipette solution (‘intracellular solution’) CsCl 125 mmol/l NaCl 8 mmol/l 1 mmol/l CaCl2 EGTA 10 mmol/l 2 mmol/l Na2ATP MgATP 3 mmol/l 0.1 mmol/l Na2GTP HEPES 10 mmol/l pH 7.2 (with CsOH) This solution should be filtered through a sterile filter before use. Transfer an aliquot of cell-containing solution to the bath which is positioned on the stage of an inverted microscope or use coverslips with cells grown on top as bath ground. Perfuse the bath at a constant rate of 1 ml/min at either room temperature (21–24 ºC) or at 37 ºC as desired. Adjust the amplifiers and compensate for series resistance or capacity. If SEVC amplifiers are used adjust the switching frequency to values of about 25–30 kHz. Make sure that the command potential is reached within 3–5 ms with an accuracy of =5 mV, even when large currents are recorded. Find a cell pair (use a 40¶ objective with 10¶ eyepieces and phase contrast) and position electrodes in the direct vicinity of the cells. Establish a gigaseal (seal resistance should exceed 5 GX) for one cell, perform the breakin and achieve the whole cell configuration by applying a sharp suction pulse. Thereafter try to establish a gigaseal and afterwards a whole-cell configuration in the other cell. 3–5 min after establishing the whole-cell configuration the experiment is started. Sampling frequency is adjusted to 10 kHz and the obtained signal is low-pass filtered at 1 kHz. During the recordings 1 mmol/l BaCl2 is added to the external solution. One can use either a symmetrical or an asymmetrical protocol. First both cells are clamped to Ö40 mV holding potential in order to inactivate the sodium current. Thereafter, one cell is clamped to potential ranging from Ö90 to +10 mV for 200 ms (pulse duration; asymmetrical protocol). Thereby, a transcellular voltage difference of ×50 mV can be applied

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Table 4. Dyes used for investigation of dye coupling Dye

Excitation

Emission wavelength (nm)

Molecular weight

Lucifer yellow Procion yellow 2,7-Dichlorofluorescein

430 488 513

535 530 532

457 697 691

and the current measured in the nonpulsed cell can be taken as the gap junctional current [Spray et al., 1981; Weingart, 1986]. Gap junction conductance can be calculated as the slope of the current-voltage relationship by linear regression analysis, if the relationship is linear. By addition of the currents flowing in the resting and the pulsed cell, the sarcolemmal current in the pulsed cell can be calculated as a current-voltage relationship [Weingart, 1986] and the input resistance can be estimated from the chord conductance between Ö80 and Ö40 mV. Measurements should be done in both cells alternatively. For a symmetrical protocol both cells are clamped to the common holding potential of Ö40 mV. Thereafter, both cells are pulsed equally but with opposite polarity. Thus, cell 1 is clamped to Ö50 mV and cell 2 to Ö30 mV, then to Ö60 and Ö20 mV, respectively, and so forth. If voltage dependence is to be investigated the pulse duration must be enhanced to values of 1–2 s or even more, and transjunctional voltages of up to 100 mV have to be applied. A common problem with gap junction measurements is a rundown of gj in these preparations, for example in neonatal rat heart cells Schmilinsky-Fluri et al. [1990] found a decrease in gj of 16.4% in 6 min which could be antagonized by addition of a phospholipase inhibitor, 20 lmol/l bromophenacyl bromide, to 1.8% within 6 min. They suggested that endogenous arachidonic acid is involved in spontaneous uncoupling. Others favored a washout of ATP and cyclic nucleotides as a possible cause and prevented their preparations from spontaneous uncoupling by addition of ATP, GTP or cAMP to the pipette solution [Mu¨ller et al., 1997a, b].

8.7 Dye-Coupling Studies A technique often used for the investigation of gap junctions is the injection of lucifer yellow (LY). However, it should be noted that dye coupling and electrical coupling can differ from each other in certain situations. For dye-coupling studies, LY is dissolved in water at concentrations of 3–5%. The cell is penetrated with a LY-filled pipette and stained by application of constant hyperpolarizing current pulses of 4–10 nA (1 s pulse duration, 0.5 Hz frequency) for 1–2 min. LY-filled pipettes exhibit higher resistance than 3 mol/l KCl electrodes and give unstable recordings. Alternatively, LY can be dissolved in 3 mol/l LiCl. If LY is used with the patch-clamp technique, LY is dissolved in the standard pipette solution at a concentration of about 0.1–0.5%. After establishing the whole cell configuration, the

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cell is clamped to negative potential for 1–5 min in order to increase the intracellular dye concentration. Staining of the injected cell and the adjacent cells can be observed directly using an epifluorescence microscope (table 4). The excitation light is emitted from a mercury highpressure lamp and filtered by a band-pass excitation filter (BP 450–490). The light then passes a dichroic mirror (FT 510) which serves as a beam splitter. Light with wavelengths of =510 nm is selectively reflected by the mirror, whereas longer wavelengths pass it. Thereby excitation light of 450–510 nm reaches the specimen and excites the dye. The light emitted by the dye passes the beam splitter and is filtered by a long-pass emission filter (LP 520) which enables wavelengths of ?520 nm to pass. It is necessary to use objectives with the microscope which are designed for high UV light transmission (e.g. Neofluar, Zeiss).

Acknowledgements I greatly thank Dr. Aida Salameh for the superior help with the figures, Mrs. Michaela Gottwald for intensive corrections and help with the search for literature, Mrs. Kathi Kru¨semann for taking the histological photographs, and Mr. Rajiv Grover for language revision.

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Wang HZ, Li J, Lemanski LF, Veenstra RD: Gating of mammalian cardiac gap junction channels by transjunctional voltage. Biophys J 1992;63:139–151. Warner A, Clements DK, Parikh S, Evans WH, DeHaan RL: Specific motifs in the external loops of connexin proteins can determine gap junction formation between chick heart myocytes. J Physiol (Lond) 1995;488:721–728. Weidmann S: The electrical constants of Purkinje fibers. J Physiol (Lond) 1952;118:348–360. Weidmann S: Passive properties of cardiac fibers; in Rosen MR, Janse MJ, Wit AL (eds): Cardiac Electrophysiology. A Textbook. Mount Kisco, Futura, 1990, pp 29–35. Weingart R: The permeability to tetraethylammonium ions of the surface membrane and the intercalated disks of sheep and calf myocardium. J Physiol (Lond) 1974;240:741–762. Weingart R: The actions of ouabain on intercellular coupling and conduction velocity in mammalian ventricular muscle. J Physiol (Lond) 1977;264:341–365. Weingart R: Electrical properties of the nexal membrane studied in rat ventricular cell pairs. J Physiol (Lond) 1986;370:267–284. Weingart R, Maurer P: Cell-to-cell coupling studied in isolated ventricular cell pairs. Experientia 1987; 43:1091–1094. Weingart R, Maurer P: Action potential transfer in cell pairs isolated from adult rat and guinea pig ventricles. Circ Res 1988;63:72–80. Werner R, Levine E, Rabadan-Diehl C, Dahl G: Formation of hybrid cell-cell channels. Proc Natl Acad Sci USA 1989;86:5380–5384. Werner R, Rabadan-Diehl C, Levine E, Dahl G: Affinities between connexins; in Hall JE, Zampighi GA, Davis RM (eds): Gap Junctions. Progress in Cell Research, vol 3. Amsterdam, Elsevier, 1993, pp 21–24. White RL, Doeller JE, Verselis VK, Wittenberg BA: Gap junctional conductance between pairs of ventricular myocytes is modulated synergistically by H+ and Ca++. J Gen Physiol 1990;95:1061–1075. White RL, Spray DC, Campos de Carvalho AC, Wittenberg BA, Bennett MVL: Some electrical and pharmacological properties of gap junctions between adult ventricular myocytes. Am J Physiol 1985;249:C447–C455. White TW, Bruzzone R, Goodenough DA, Paul DL: Mouse Cx50, a functional member of the connexin family of gap junction proteins, is the lens fiber protein MP70. Mol Biol Cell 1992;3:711–720. Wiener N, Rosenblueth A: The mathematical formulation of the problem of conduction of impulses in a network of connected excitable elements, specifically in cardiac muscle. Arch Inst Cardiol Mex 1946;16:205–265. Wier WG, Cannell MB, Berlin JB, Marban E, Lederer WJ: Cellular and subcellular heterogeneity of [Ca2+]i in single heart cells revealed by Fura-2. Science 1987;235:325–328. Wilde AAM, Escande D, Schumacher CA, Thuringer D, Mestre M, Fiolet JWT, Janse MJ: Potassium accumulation in the globally ischemic mammalian heart. A role for the ATP-sensitive potassium channel. Circ Res 1990;67:835–843. Wilders R, Jongsma HJ: Limitations of the dual voltage clamp method in assaying conductance and kinetics of gap junction channels. Biophys J 1992;63:942–953. Willecke K, Hennemann H, Dahl E, Jungbluth S: The mouse connexin family; in Hall JE, Zampighi GA, Davis RM (eds): Gap Junctions. Progress in Cell Research, vol.3. Amsterdam, Elsevier, 1993, pp 33–37. Willecke K, Heynkes R, Dahl E, Stutenkemper R, Hennemann HJ, Jungbluth S, Suchyna T, Nicholson BJ: Mouse connexin37: Cloning and functional expression of a gap junction gene highly expressed in lung. J Cell Biol 1991;114:1049–1057. Wit AL, Janse MJ: The ventricular arrhythmias of ischemia and infarction. Electrophysiological mechanisms. Futura Publishing Company, Mount Kisco, 1992, pp 1–160. Wojtcak J: Contractures and increase in internal longitudinal resistance of cow ventricular muscle induced by hypoxia. Circ Res 1979;44:88–95. Wu J, Corr PB: Influence of long chain acylcarnitines on the voltage-dependent calcium current in adult ventricular myocytes. Am J Phyisol 1992;263:H410–H417. Wu J, McHowat J, Saffitz JE, Yamada KA, Corr PB: Inhibition of gap junctional conductance by long chain acylcarnitines and their preferential accumulation in junctional sarcolemma during hypoxia. Circ Res 1993;72:879–889.

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Yamada KA, McHowat J, Yan GX, Donahue K, Peirick J, Kle´ber AG, Corr PB: Cellular uncoupling induced by accumulation of long-chain acylcarnitine during ischemia. Circ Res 1994;74:83–95. Yamamoto T, Ochalski A, Hertzberg EL, Nagy JL: LM and EM immunolocalization of the gap junctional protein connexin 43 in rat brain. Brain Res 1990;508:313–319. Yancey SB, Edens JE, Trosko JE, Chang C, Revel JP: Decreased incidence of gap junctions between Chinese hamster V-79 cells upon exposure to the tumor promotor 12-O-tetradecanoyl phorbol-13acetate. Exp Cell Res 1982;139:329–340. Yang H, Muller-Borer BJ, Johnson TA, Sandiford DR, Lemasters JJ, Cascio WE: Na/HCO3 cotransport and Na/H exchange contribute to the rate of cell-to-cell electrical uncoupling in ischemic ventricular myocardium (abstract 3472). Circulation 1996;94(suppl I):593. Zampighi G: Gap junction structure; in DeMello WC (ed): Cell-to-Cell Communication. New York, Plenum Press, 1987, pp 1–28. Zhang JT, Nicholson BJ: Sequence and tissue distribution of a second protein of hepatic gap junctions, Cx26, as deduced from its cDNA. J Cell Biol 1989;109:3391–3401. Zimmer PB, Green RC, Evans HW, Gilula NB: Topological analysis of the major protein in isolated intact rat liver gap junctions and gap junction derived single membrane structures. J Biol Chem 1987;262:7751–7763.

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Appendix

One-letter code of amino acids, three-letter code and codons (5–3) coding for the amino acid Amino acids with apolar side chains A Ala Alanine V Val Valine L Leu Leucine I Ile Isoleucine P Pro Proline F Phe Phenylalanine W Trp Tryptophane M Met Methionine

GCU, GCC, GCA, GCG GUU, GUC, GUA, GUG CUU, CUC, CUA, CUG, UUA, UUG AUU, AUC, AUA CCU, CCC, CCA, CCG UUU, UUC UGG AUG

Amino acids with uncharged polar side chains G Gly Glycine S Ser Serine T Thr Threonine C Cys Cysteine Y Tyr Tyrosine N Asn Asparagine Q Gln Glutamine

GGU, GGC, GGA, GGG AGU, AGC, UCU, UCC, UCA, UCG ACU, ACC, ACA, ACG UGU, UGC UAU, UAC AAU, AAC CAA, CAG

acid amino acids (negatively charged at pH 6) D Asp Aspartatic acid GAU, GAC E Glu Glutamic acid GAA, GAG basic amino acids (positively charged at pH 6) K Lys Lysine AAA, AAG R Arg Arginine CGU, CGC, CGA, CGG, AGA, AGG H His Histidine CAU, CAC Stop codons: UAA, UAG, UGA One-letter code for nucleic acids is: A>adenine; G>guanine; C>cytosine; U>uracil.

140

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List of Suppliers of Specialized Items

Axon Instruments 1101 Chess Drive Foster City, CA 94404 (USA) Tel. +1 415 571 9400 Fax +1 415 571 9500 In Germany: 0130 81 0458 In Switzerland: 046 05 7323 (electrophysiological equipment, patch clamp amplifiers, switch clamp amplifiers) Becton-Dickinson Labware (Falcon) 1950 Willaim Drive Oxnard, CA 93030 (USA) Becton Dicknson GmbH Postfach 101629 D–69006 Heidelberg (Germany) Tel. +49 6221 3050 Fax +49 6221 303609 (cell culture equipment) Biermann GmbH (DPC) Hohe Strasse 4-8 D–61231 Bad Nauheim (Germany) Tel. +49 6032 99400 Fax +49 6031 994200 (antibodies)

Fax +49 551 5068666 550 N. Reo Street #101 Tampa, FL 33609 (USA) Tel. +1 813 287 8815 Fax +1 813 282 1936 (labware, SDS-PAGE equipment) Boehringer Mannheim Sandhofer Strasse 116 D–68305 Mannheim (Germany) Tel. +49 621 7590 Fax +49 621 759 2890 (biochemistry, signal transduction reagents, immunochemistry) Chemicon 28835 Single Oak Drive Temecula, CA 92590 (USA) Tel. +1 909 676 8080 Fax +1 909 676 9209 (antibodies) Clark Electromedical Instruments PO Box 8 Pangbourne, Reading RG8 7HU (UK) Tel. +44 1734 843888 Fax +44 1734 845374 (electrophysiological equipment)

Biologic Rue de l’Europe 1 F–38640 Claix (France) Tel. +33 76 986831 Fax +33 76 986909 (electrophysiological equipment, pullers, patch clamp amplifiers)

Dianova Postfach 301250 D–20305 Hamburg (Germany) Tel. +49 40 45067 0 Fax +49 40 45067 390 (antibodies)

Biometra Rudolf-Wissell Strasse 30 D–37079 Go¨ttingen (Germany) Tel. +49 551 506860

FMI Fo¨hr Medical Instruments In der Grube 13 D–64342 Seeheim/Ober-Beerbach (Germany)

141

Tel. +49 6257 962260 Fax +49 6257 962262 (electrophysiological equipment, pullers, patch clamp amplifiers) Gibco/BRL Life Technologies Dieselstrasse 5 D–76344 Eggenstein (Germany) Tel. +49 130 830902 or: Gibco Ltd PO Box 35 Paisley PA3 4EF (UK) (cell culture media, biochemica and equipment) Greiner Labortechnik Maybachstrasse 2 D–72636 Frickenhausen (Germany) (plastic cell culture flasks, cell culture equipment) Hameg Kelsterbacher Strasse 15–19 D–60528 Frankfurt/Main (Germany) Tel. +49 69 678050 Fax +49 69 6780513 (oscilloscopes) Heka Elektronik Wiesenstrasse 71 D–67466 Lambrecht (Germany) Tel. +49 6325 8036 Fax +49 6325 8039 (electrophysiological equipment, pullers, patch clamp amplifiers) Heraeus Instruments GmbH Postfach 1563 D–63405 Hanau (Germany) Tel. +49 6181 35 413 Fax +49 6181 35 739 (cell culture equipment, incubators, benches)


Hewlett-Packard Rothebu¨hlstrasse 81 D–70197 Stuttgart (Germany) Tel. +49 711 61965 0 Fax +49 711 61965 50 (oscilloscopes, recorders) Immunotech Luminy case 915 F–13288 Marseille Cedex 9 (France) Postfach 101526 D–20010 Hamburg (Germany) Tel. +49 40 322180 Fax +49 40 323969 (antibodies) Integra Biosciences Tecnomara GmbH Ruhberg 4 D–35461 Fernwald (Germany) Tel. +49 6404 8090 (cell culture equipment) Leica Vertrieb GmbH Lilienthalstrasse 39–45 D–64606 Bensheim (Germany) Tel. +49 6251 136 0 Fax +49 6251 136 155 (microscopes and equipment) List Elektronic Pflungsta¨dter Strasse 18–20 D–64297 Darmstadt (Germany) Tel. +49 6151 56000 Fax +49 6151 56060 (electrophysiological equipment, pullers, patch clamp amplifiers) Luigs & Neumann Boschstrasse 19 D–40880 Ratingen (Germany) Tel. +49 2102 4420 35 Fax +49 2102 4420 36 (micromanipulators and setups)

142

Ma¨rzhaüser Wetzlar GmbH & Co KG An den Fichten 35 D–35579 Wetzlar (Germany) Tel. +49 6441 9116 0 Fax +49 6441 9116 40 (micromanipulators) Merck Frankfurter Strasse 250 D–64293 Dramstadt (Germany) Tel. +49 6151 72 0 Fax +49 6151 72 2000 (biochemica) Millipore GmbH Hauptstrasse 87 D–65760 Eschborn (Germany) Tel. +49 6196 4940 Fax +49 6196 43901 or: Millipore Corporation 8 Ashby Road Bedford, MA 01730 (USA) (filter technique, cell biology) Narishige Unit 7 Willow Business Park, Willow Way London SE2 64QP (UK) Tel. +44 181 699 8282 Fax +44 181 699 8299 (electrophysiological equipment, pullers, micromanipulators) Nikon Tiefenbroicher Weg 25 D–40472 Du¨sseldorf (Germany) Tel. +49 211 9414 0 Fax +49 211 9414 330 (microscopes and equipment) NPI electronic Ha¨ldenstrasse 62 D–71732 Tamm Tel. +49 7141 60 1534 Fax +49 7141 60 1266 (electrophysiological equipment, patch clamp amplifiers, switch clamp amplifiers)


Nunc GmbH Hagenauer Strasse 21a D–65203 Wiesbaden-Biebrich (Germany) Tel. +49 611 67095 Fax +49 611 607348 or: 2000 Aurora Road Naperville, IL 60566 (USA) (cell culture equipment) Olympus Optical GmbH & Co. Wendenstrasse 14-19 D–20097 Hamburg (Germany) Tel. +49 40 23773 0 Fax +49 40 23773 647 (microscopes, microforges and equipment) Pacer Scientific Instruments 5649 Valley Oak Drive Los Angeles, CA 90068 (USA) Tel. +1 213 462 0636 Fax +1 213 462 1430 (recorder, micromanipulators, glass capillaries, puller, stimulators, amplifiers) Sarstedt Postfach 1220 D–51582 Nu¨mbrecht (Germany) Tel. +49 2293 3050 (cell culture equipment, plastic culture flasks) Sartorius AG D–37070 Go¨ttingen (Germany) Tel. +49 551 3080 (filters for cell cultures, labware) Science Products Hofheimer Strasse 63 D–65719 Hofheim (Germany) Tel. +49 6192 5046 Fax +49 6192 5053 (electrophysiological equipment, pullers, patch clamp amplifiers)

143

Sigma Chemie Gru¨nwalder Weg 30 D–82039 Deisenhofen (Germany) Tel. +49 0130 5155 Fax +49 0130 6490 or: Sigma Chemical Co. PO Box 14508 St. Louis, MO 63178 (USA) (chemicals, antibodies, cell culture equipment, labware) Sutter Instruments 40 Leveroni Court Novato, CA 94949 (USA) Tel. +1 415 883 0128 Fax +1 415 883 0572 (electrophysiological equipment, pullers, patch clamp amplifiers) Tektronix Colonia Allee 11 D–51067 Ko¨ln (Germany) Tel. +49 221 96969 0 Fax +49 221 96969 362 (oscilloscopes)

Warner Instrument Corp. 1125 Dixwell Avenue Hamden, CT 06514 (USA) Tel. +1 203 776 0664 Fax +1 203 776 1278 (glass, AD/DA converter, amplifiers and equipment) WPI World Precision Instruments Harry Fein Liegnitzer Strasse 15 D–10999 Berlin (Germany) Tel. +49 30 618 8845 Fax +49 30 618 8670 (microscopes, oscilloscopes and electrophysiological equipment) Carl Zeiss Jena Tatzendpromenade 1a D–07740 Jena (Germany) Tel. +49 3641 64 2420 Fax +49 3641 64 3140 (microscopes, micromanipulators and equipment)

The items which may be of interest for gap junction research as outlined in the book are given in brackets. This is, however, not the entire product list of the supplier. It was not possible to incorporate all companies which supply items in the various fields of research. This is a list of suppliers mentioned somehow in this book and is not intended to represent a complete list of suppliers for cell culture techniques, electrophysiology, biochemistry and labware.


144

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Subject Index

AAP10, gap junction uncoupling 102, 104, 105 Acetic acid, gap junction uncoupling 94 Acetylcholine, gap junction channel regulation 46 Aconitine, gap junction uncoupling 95, 96 Action potential propagation changes in arrhythmia 12 coupling effects on transverse and longitudinal propagation 5, 6 effects of nonuniformity 4, 5 Acute cardiac disease, effects on gap junctions 73–78 Acylcarnitines, gap junction uncoupling 74–76, 94 Adrenaline, gap junction channel regulation 47, 99 Aging, effects on gap junctions 88 Angiotensin II, gap junction uncoupling 100, 101 Anisotropic ratio, regional variations 6 Anisotropic reentry 9, 11 Antiarrhythmic peptides 101, 102, 104, 105 Arachidonic acid, gap junction channel regulation 45, 46, 93, 94 Arrhythmia effects on gap junctions 83, 84 types in ischemia 10, 11, 74 Arrhythmogenic substrate, definition 1, 2 Assembly, gap junction channel 64–69

ATP, gap junction channel regulation 44, 45 Atrioventricular bundle, gap junction distribution 28, 30 Atrioventricular node, gap junction distribution 27, 29, 30 Atrioventricular reentry 9 Atrium, gap junction distribution 29 Bundle branches, gap junction distribution 30 Cable properties, muscle fibers 3, 4 Caffeine, effect on uncoupled gap junctions 96 Calcium, gap junction channel regulation 37, 38, 40–43 cAMP, see Cyclic AMP Capacitive coupling 2, 3 Carbachol, gap junction uncoupling 98, 99 Carbon dioxide, see pCO2 Cardiac arrhythmia suppression trial (CAST), proarrhythmic risk of antiarrhythmic drugs 1 Cardiac myocyte culture embryonic chick cardiomyocytes 108, 109 neonatal rat cardiomyocytes 106–108 fibroblast-myocyte gap junctions 33 isolation from guinea pig 109, 110 shape effects on propagation 7, 8

145

CAST, see Cardiac arrhythmia suppression trial Cell culture electrophysiology, systems for study 50 embryonic chick cardiomyocytes 108, 109 neonatal rat cardiomyocytes 106–108 Chagas disease, effect on gap junctions 84–86 Chronic ischemic heart disease, effects on gap junctions 11, 79–81 Connexins assembly 66–69 assembly of gap junctions 64–66, 68 Cx40 gene mutations 87 Cx43 gene mutation in defective heart development 86, 87 regulation 71, 72 isolation 114–116 physical properties 14, 15 isoform expression in gap junctions 6, 13 multiple protein channels 34 posttranslational modifications 14–16, 40, 66, 69 sequences 20, 21 single-channel conductance Cx26 channels 61 Cx37 channels 60, 61 Cx40 channels 59, 60 Cx43 channels 59 Cx45 channels 60 species variability 23, 24 staining and distribution 27–31, 110–114 structure 13, 14 synthesis 64–66, 68, 69, 71, 72 transjunctional voltage sensitivity 55–57 turnover 68, 69 types 13, 19–23 Coronary vasculature chronic ischemic heart disease, effect on gap junctions 81 gap junction distribution 31 Current components 52

Subject Index

equations 51, 52 voltage relationships in gap junction channels 52–54 Cyclic AMP (cAMP), gap junction channel regulation 35–38, 69, 70 Decanoic acid, gap junction uncoupling 91 Defective heart development, effects on gap junctions 86, 87 Degradation, gap junction channel 68, 69 Distribution, see Gap junction distribution Double-cell voltage-clamp technique 50, 51, 116–119 Dye-coupling studies 119, 120 Electric field coupling 3 Electron microscopy, gap junction distribution 25, 26, 34 Electrophysiology, gap junction channels cell systems for study 50 connexins Cx26 single-channel conductance 61 Cx37 single-channel conductance 60, 61 Cx40 single-channel conductance 59, 60 Cx43 single-channel conductance 59 Cx45 single-channel conductance 60 transjunctional voltage sensitivity 55–57 current components 52 equations 51, 52 voltage relationships 52–54 double-cell voltage-clamp technique 50, 51, 116–119 intercellular resistance 52, 54, 58, 59 ion permeability 61, 62 open probability of a single channel 57 states of single-channel conductance 50 types of channels 50 voltage sensitivity of channels 56, 57 Enalapril, effect on uncoupled gap junctions 100, 101 Endothelial cells, gap junction distribution 31 Ethrane, gap junction uncoupling 90

146

Excitation, transfer between cardiac cells 2, 3 FGF-2, see Fibroblast growth factor-2 Fibroblast growth factor-2 (FGF-2) gap junction channel regulation 48, 71 release in chronic ischemic heart disease 79 Gap junction channel assembly 64–69 cardiac disease-induced changes acute cardiac disease 73–78 aging 88 arrhythmia 83, 84 chronic ischemic heart disease 79–81 defective heart development 86, 87 heart failure 82, 83 infective heart disease 84–86 closure mechanism 18 clustering 17 degradation 68, 69 developmental expression 63, 66 electrophysiology, see Electrophysiology, gap junction channels functions, overview 13, 25, 48–50 new channel formation, kinetics 63, 64 regulation, see specific regulators structure 13, 16, 17 synthesis 64–72 uncoupling drugs, see specific drugs effects in heart disease 77, 78, 89, 90 Gap junction distribution atrioventricular bundle 28, 30 atrioventricular node 27, 29, 30 atrium 29 bundle branches 30 chronic ischemic heart disease effects 89, 81 connexin staining 27–31, 110–114 coronary vasculature 31 effects on conduction 9, 10, 25 electron microscopy 25, 26, 34 endothelial cells 31 heart failure effects 82

Subject Index

laser scanning confocal microscopy 27, 34 multiple connexin channels 34 myocyte-fibroblast junctions 33 Purkinje fibers 30 sinoatrial node 29 topology 26, 27 ventricular myocardium 30, 31, 34 Halothane, gap junction uncoupling 91 Heart failure, effects on gap junctions 82, 83 Heptanol, gap junction uncoupling 90 Hypertension, effect on gap junctions 83 Immunostaining connexins 27–31 protocol 110–114 Infective heart disease, effects on gap junctions 84–86 Ion permeability, gap junction channels 61, 62 Ischemia associated arrhythmias 10, 11 gap junction changes acute cardiac disease 73–78 chronic ischemic heart disease 11, 79–81 tissue properties in healing phase 11 Laser scanning confocal microscopy, gap junction distribution 27, 34 Leading circle concept 8 Magnesium, gap junction channel regulation 44 Myocyte, see Cardiac myocyte Noradrenaline, gap junction channel regulation 47, 99 Octanol, gap junction uncoupling 90 Oleic acid, gap junction uncoupling 92, 93 Ouabain, gap junction uncoupling 95 Palmitoleic acid, gap junction uncoupling 91

147

pCO2, gap junction channel regulation 43 Permeability, gap junction channels to ions 61, 62 pH, gap junction channel regulation 42, 43 Phospholipase C (PLC), gap junction channel regulation 38 Phosphorylation, gap junction channel regulation 40, 97 PKA, see Protein kinase A PKC, see Protein kinase C PKG, see Protein kinase G PLC, see Phospholipase C Potassium efflux, acute cardiac disease 73, 74 Propagation velocity, ischemia effects 75, 76, 90 Prophylaxis, proarrhythmic risk of antiarrhythmic drugs 1, 2 Propionic acid, gap junction uncoupling 94, 95 Protein kinase A (PKA), gap junction channel regulation 35–39 Protein kinase C (PKC), gap junction channel regulation 38, 39, 46, 70, 97–100

Subject Index

Protein kinase G (PKG), gap junction channel regulation 39, 40 Purkinje fibers, gap junction distribution 30 Resistance, gap junction channels 52, 54, 58, 59 Sinoatrial node, gap junction distribution 29 Sodium, gap junction channel regulation 44 Strophanthidin, gap junction uncoupling 95 Synthesis, gap junction channel 64–72 Temperature, gap junction channel regulation 35 Tyrosine kinase, gap junction channel regulation 40, 70, 71 Uncoupling, gap junction channel drug induction 90–105 effects in heart disese 77, 78, 89, 90 Ventricular myocardium, gap junction distribution 30, 31, 34

148

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