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

Cardiac Gap Junctions

Physiology, Regulation, Pathophysiology and Pharmacology
S. Dhein, Cologne
23 gures and 3 tables, 1998
...........................
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.
Copyright 1998 by S. Karger AG, P.O. Box, CH4009 Basel (Switzerland)
Printed in Switzerland on acid-free paper by Reinhardt Druck, Basel
ISBN 3805565674
IV
Dedicated to Aida
V
...........................
Contents
Foreword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XI
1 Introduction: Cellular Coupling, Cardiac Activation Patterns and
Arrhythmia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2 Structure and Diversity of Gap Junction Channels . . . . . . . . . . 13
3 Distribution of Gap Junctions in the Heart . . . . . . . . . . . . . . 25
4 Function and Physiology of Gap Junction Channels . . . . . . . . . 35
5 Regulation of Gap Junction Expression, Synthesis and Assembly . 63
6 Gap Junctions in Cardiac Disease . . . . . . . . . . . . . . . . . . . . 73
7 Pharmacological Interventions at Gap Junctions . . . . . . . . . . . 89
8 Methods for Investigation of Gap Junctions . . . . . . . . . . . . . . 106
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
List of Suppliers of Specialized Items . . . . . . . . . . . . . . . . . . 141
Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
VII
...........................
Foreword
When I started as a novice in the eld 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 bers and muscular trabeculae
were the preferred cardiac preparations. These multicellular preparations were
suitable to perform cable analyses and diusion studies. At that time, my
mentor, Silvio Weidmann, had already accomplished his elegant functional
studies.
The subsequent progress in the eld was prompted largely by the develop-
ment 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 ow. 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 molecu-
lar 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 relation-
ship 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
IX
...........................
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 inuencing the adjacent cells. In the chronic phase of
cardiac disease the distribution of various gap junction isoforms can change,
thereby altering the tissues 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 eld
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 eld of research. Therefore, the rst
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 de-
scribed 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 nal chapter is especially written for people who are interested in entering
this fascinating eld 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-coup-
ling assays are described with practical protocols. At the end of the book a
list of suppliers of specialized items, such as certain ampliers, antibodies etc.,
is given.
Stefan Dhein
Cologne, April 1997
XI
1
...........................
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 understand-
ing 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 (ecainide) 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 con-
centrations. This has been dened as the proarrhythmic risk of antiarrhythmic
drugs. First, it was conned 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 signicant
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 nally 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 nding 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 brosis,
aneurysm, the border zone between normal and ischemic or infarcted tissue,
scars, diuse myocardial injury in cardiomyopathy or the chronic alterations
induced by myocarditis, and furthermore, accessory pathways or variations in
the specic cardiac conduction system. These anatomic or pathologic altera-
1
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 nally cause
arrhythmia.
In consequence, the goal of many studies was to dene the changes
responsible for arrhythmogenesis on the tissue or whole organ level. Many
investigators sought for new approaches to arrhythmogenesis and antiarrhyth-
mic agents. Especially safe antiarrhythmic agents for use in prophylactic treat-
ment are searched for.
In the course of this development focusing on the arrhythmogenic sub-
strate 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 stretch-
ing, 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 trans-
mitter, since the K
+
euxing during an action potential will diuse rapidly
in the bulk interstitial uid 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 identied as gap junction chan-
nels. An early argument in support of the concept of low-resistance pathways
was that the length constant (measured in cardiac muscle bundles by extracel-
lular 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
eld coupling have been proposed as alternative mechanisms of electrical
transmission. Capacitive coupling according to Sperelakis [1979] means that
a capacitive current ows through a capacitance (the membrane is a capacitor
and the action potential is an alternating current AC signal) that acts to couple
2 1 Introduction
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 eld coupling [Sperelakis and
Mann, 1977] means the induction of an action potential in the post-cell by
the electrical eld 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 re a fraction of a millisecond earlier than
the surface membrane, which was necessary for eective coupling. However, at
present it is uncertain what the contribution of electrical eld 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 eld coup-
ling [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 eld coupling or capacitive
coupling. The mass of activated tissue might be a determining feature. The
composition of the interstitial uid, 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 mecha-
nism for transmission of excitation is coupling via the gap junction channels.
Considering the passive electrical properties of the tissue means rst of
all to consider the properties of a muscle bundle, i.e. the passive cable properties.
Muscle bers 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 V
x
>V
x0
(expx/) with the length constant
>Y(r
m
/r
i
) (r
m
>membrane resistance, r
i
>internal longitudinal resistance);
the input resistance at x>0 can be described as r
input
>V
x0
/I>r
i
. Taking the
ber radius into account, the specic membrane resistance R
m
equals 2ar
m
[cm
2
] and specic internal resistance R
i
>r
2
r
i
. With the specic membrane
3 1 Introduction
capacitance the time constant is described as >C
m
R
m
. In a multicellular
preparation with parallel running bers the longitudinal resistance of the
extracellular space r
o
also has to be considered. For these conditions is
reected by >Y(r
m
/[r
i
+r
o
]) and the conduction velocity depends on
>Y(1/{T
foot
C
m
[r
i
+r
o
]}). However, this cable theory, originally formulated for
nerve axons [Hodgkin and Rushton, 1946] and later on for Purkinje bers
[Weidmann, 1952], is based on the assumption of continuity of the cable. In
consequence of these assumptions, passive membrane properties were consid-
ered to be of minor importance for the pathophysiology of conduction distur-
bances and the basic mechanisms were ascribed to membrane ionic properties
and their regional dierences.
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 bers than in the transverse direction due
to higher intercellular resistance perpendicular to the ber axis. In addition,
on a microscopic basis propagation is discontinuous and the inhomogeneous
and anisotropic distribution of the cellular connections inuence action poten-
tial upstroke and the safety factor of propagation [Spach and Dolber, 1990].
The dierence between the two forms of anisotropy, i.e. uniformversus nonuni-
form, 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 reection of discontinuous propagation and each of the
multiple negative peaks represent the activation of a small group of bers. 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 aniso-
tropy due to predominant uncoupling of side-to-side connections with increas-
ing age [Spach and Dolber, 1986, 1990]. What are the consequences of non-
uniformity for action potential propagation? In bers with tight electrical
coupling, simulated extrasystoles with the shortest interval leading to a propa-
gated response lead to a progressive reduction in conduction velocity in all
directions (i.e. in parallel to the reduced sodium channel availability). In con-
trast, in nonuniform tissue the earliest premature beat leads to dissociated
microscopic longitudinal propagation, i.e. the large biphasic waveformchanges
4 1 Introduction
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 non-
uniform anisotropic tissue can induce conduction block in longitudinal direc-
tion 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 ndings that the
microreentry was solely based on discontinuous anisotropic propagation. The
safety factor for longitudinal propagation is reduced when sodiumconductance
is decreased as the consequence of early premature beats. Under these condi-
tions the discontinuities in nonuniform anisotropic tissue can cause longitud-
inal 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, slowconduction does not necessarily mean depressed conduction [Spach
and Dolber, 1990].
What are the eects 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 ndings of Spach and coworkers considered above, the
authors suggested that uncoupling can have opposite directional eects 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 re-
duced sodium channel availability especially in nonuniform anisotropic tissue,
and under these conditions reentrant arrhythmia can be initiated due to discon-
tinuous propagation, whereas transverse propagation is more sensitive to un-
5 1 Introduction
coupling. However, it should be taken into account that heptanol does not
specically block gap junctions and that under most pathophysiological condi-
tions, 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 dicult 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 inuenced by the intercellular coupling. At least four features have
to be considered. (1) Cardiac cells express dierent gap junction proteins (so-
called 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 dierent with regard to their biophysical properties.
In various parts of the heart the content of each of these isoforms is dierent.
(2) There is a highly distinctive three-dimensional spatial distribution of inter-
cellular connections with dierent patterns in dierent parts of the heart.
(3) Furthermore, it has been shown that a single cardiomyocyte can express
dierent isoforms of connexins [Satz 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 specic 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 dierent 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? Satz et al. [1994] showed that
in the crista terminalis each cell is connected to 6.41.7 other cells whereas
in the ventricle each cell is connected to 11.32.2 other myocytes. In addition,
in the crista terminalis the gap junctions were conned 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 eective length to width ratio of a ventri-
cular cell is reduced from 6:1 to 3.4:1 [Satz 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, how-
ever, 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
6 1 Introduction
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
Satz, 1991] (see chapter 6). As a consequence, a wavefront travelling trans-
verse to the ber axis becomes slowed specically 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 pathophysiolog-
ical alterations in the activation pattern, and thus are an important determining
factor in arrhythmogenesis. These factors have long been neglected, cardiovas-
cular 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 nite number of 10
*
10-m 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 S
(combined plicate junction). They found that during longitudinal propagation
the upstroke velocity of the local action potential V
max
was lowest at the
proximal end of the cell, increased to its maximum at the distal fourth and
decreased distally. During transverse propagation, higher V
max
as well as rapid
intracellular conduction with variable intracellular pathways was observed.
At the end of some myocytes higher V
max
was found for transverse propagation.
The charge elicited by the fast sodium current was inversely related to V
max
.
The surrounding cellular network exhibited a strong modulating inuence
on gap junction delay, V
max
and sodium current. Coupling the cells to their
surrounding resulted in a decrease in V
max
, 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
7 1 Introduction
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 V
max
have
been observed in anisotropically grown cardiomyocyte cultures if major discon-
tinuities existed using voltage-sensitive dyes [Fast and Kleber, 1994; Fast et al.,
1996; Rohr, 1995]. In additional computer simulations these authors could
show that the dierence between longitudinal and transverse depended on the
degree of anisotropy and the pattern of gap junctions. The dierence 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 eect of longitudinal clefts on action potential
propagation. They found that such structures result in local dierences in V
max
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 reected
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 overviewon 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. Dierent 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 formof a centrally nonexcited region.
Following this concept, which is known as the leading circle concept, the
membrane potential of these central bers is held above threshold due to the
electrotonic inuence 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 3050 mm
2
is required. In 1966 Krinsky
8 1 Introduction
demonstrated in a 2-dimensional isotropic model that localized delays of
conduction within the circuit reduce the minimum perimeter of the reverber-
ator so that it may be less than the wavelength of refractoriness (>RP
*
V;
RP>refractory period, V>average conduction velocity).
This is substantially dierent from the classical reentry model requiring
a central obstacle with the reentrant circuit path length equalling the wave-
length of refractoriness (>RP
*
V) [Wiener and Rosenbluth, 1946]. Similar
forms of reentry can be initiated by S1 and S2 at dierent 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 pos-
sible in small areas of =1015 or even =2 mm
2
[Spach et al., 1988]. This
nonuniformity can be caused by either microbrosis with connective tissue
septae separating the bers 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 dierent 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 conned to the
interval between the refractory periods of longitudinal and transverse propa-
gated 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 discon-
tinuities 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
9 1 Introduction
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 bril-
lation 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 rst 24 h ventricular arrhythmias often occur.
Within the rst 30 min of ischemia two types of arrhythmia can be
distinguished: type-1a arrhythmias occur after 210 min with a peak at 56
min. They often originate from the subepicardium and the mechanism is
assumed to be associated with diastolic bridging leading to reentrant ar-
rhythmia. Besides this, non-reentrant type-1a arrhythmias can also occur which
may be due to the ow of injury current across the ischemic border causing
ectopic activity [Janse and Wit, 1989]. Type-1b arrhythmias occur later at
1230 min with a peak between 15 and 20 min and are considered to be due
to a partial recovery of dU/dt
max
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 36 h of predominantly sinus
rhythm. Thereafter, the number of ventricular ectopic beats increases. In the
subacute phase of infarction (1224 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 bers in the subendocardium. The predominant mecha-
nism has been postulated to be abnormal automaticity in these bers. These
bers exhibit an increased sensitivity for catecholamines. In some cases a
combination of focal activity and reentry in these bers may be possible.
10 1 Introduction
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 brillation. Within the infarcted
area islets of surviving myocardial cells may be found. These cells show some-
what altered electrophysiological properties with maximum diastolic potential,
action potential amplitude and duration as well as maximum upstroke velocity
being moderately reduced during the rst week after the infarction. Thereafter,
these parameters return to normal except the action potential duration. Con-
duction velocity transverse to the ber axis is reduced, while velocity parallel
to the ber is nearly normal, which may reect the loss of side-to-side connec-
tions 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 special-
ized interdigitating fibers with certain connexins forming the basis of intercellu-
lar 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 bers by strands
of connective tissue as occurring in microbrosis; (2) changes in the distribu-
tion 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.
11 1 Introduction
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 V
max
and in the time constant of the action potential foot
(
foot
). The phase-plane loops became nonlinear. Shaw and Rudy [1995] demon-
strated in a cable model, using the Luo-Rudy membrane model, that the
vulnerable window of unidirectional block depends on both membrane excit-
ability 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 ber, the model became
more sensitive to inhomogeneities in membrane excitability. As shown experi-
mentally 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 [Kleber 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).
12 1 Introduction
2
...........................
Structure and Diversity of Gap Junction
Channels
The gap junctional channel has two main functions: (a) to allow transport
of small molecules (MW=1,2001,900 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 uorescein or lucifer yellow, which are often used in dye-
coupling 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 o.
The structure of gap junction channels has been investigated employing
electron microscopy, X-ray diraction methods and molecular biology. By
these techniques it was possible to dene 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. Correspond-
ing 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 dierent isoforms (see
g. 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 con-
nexons consist of 6 polypeptide subunits, the so-called connexins, which sur-
round 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 claried. 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 dierent connexins and dierent 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 recog-
nized by regulatory protein kinases (see chapter 4) can be found. The extracellu-
lar 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 disulde
bonds, although so far there are no hints on intermolecular disulde bonds
from SDS-PAGE studies determining the molecular weight of connexins. In
contrary, in Cx43 channels it was shown that the connexon integrity is main-
tained by noncovalent bonds and that there are no intermolecular disulde
bonds [John and Revel, 1991]. Only the possibility of intramolecular disulde
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 cross-
hybridize 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 rst 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 ts well with that
obtainedfrombiochemical isolationof cardiac gapjunctionproteins by Manju-
nathandPage [1985, 1986] using SDS-PAGE(4447 kD). The dierence may be
due touncertainties withthe SDS-PAGEmethodor toaco- or posttranslational
phosphorylation as is suggested in the study by Crowet al. [1990]. Partial amino
acidsequencingstudies of the isolatedproteinrevealedconsiderablyhighhomo-
logybetweenthe foundandpredictedsequence near the Nterminus [Manjunath
et al., 1987; Nicholson et al., 1985]. From the sequencing studies it has been
14 2 Structure and Diversity of Gap Junction Channels
Fig. 1. Amino acid sequence of chicken Cx43 according to Veenstra et al. [1993]. Possible
targets for phosphorylating enzymes are underlined.
concluded that the rst methionine residue is removed posttranslationally so
that in the mature protein the rst 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 identied 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 gure 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)
15 2 Structure and Diversity of Gap Junction Channels
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 MGDWSALGKLLDKVQAYSTAGGK VWLSVLFIFRILLLGTAV ESAWGDEQS
51 AFRCNTQQPGCENVCYDKSFPISHVR FWVLQIIFVSVPTLLYLA HVFYVM
101 RKEEKLNKKEEELKVAQTDGVNVEMHLKQIEIKKFKYGIEEHGKVKMRGG
151 LLRTYIISILFKSVFEVA FLLIQWYIYGFSLSAVYTCKRDPCPHQVDCFL
201 SRPTEKT IFIIFMLVVSLVSLALNI IELFYVFFKGVKDRVKGRSDPYHAT
251 TGPLSPSKDCGSPKYAYFNGCSSPTAPLSPMSPPGYKLVTGDRNNSSCRN
301 YNKQASEQNWANYSAEQNRMGQAGSTISNSHAQPFDFPDDNQNAKKVAAG
351 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
ber [Eberth, 1866]. With modern phase contrast microscopes they can easily
be seen as shown in gure 2.
These bands were a matter of discussion for a long time until in 1954, for
the rst time, Sjo strand and Andersson used electron microscopy to investigate
intercalated disks in ultrathin osmium tetroxide-xed sections of the mouse
heart revealing that the disks were indeed transverse cell boundaries. Sub-
sequently, several investigators reproduced their nding [Lindner, 1957; Moore
16 2 Structure and Diversity of Gap Junction Channels
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
200300 A

, whereas the desmosome is a more complex and almost laminated

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
diraction 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 specic 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 diraction studies and electron microscopy. These
studies revealed the spatial structure of the channel with a total length of
approximately 100150 A

and about 52 A

in the portion within the lipid bilayer

of each side [Chen et al., 1989] and the two parallel membranes separated by
a 20 A

gap spanned by the channels subunits [Beyer et al., 1995]. The diameter
of the channel ranges between 20 and 30 A

and about 15 A

in the extracellular
half of the lipid bilayer. The inner pore appeared solvent lled, 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
three-dimensional model of the connexon with 50 A

height and 6 lobes pro-

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 scan-
ning 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 three-
dimensional model for the channel. According to these studies the gap junction
channels are arranged in clusters as shown in gure 3. A model of a single
channel is given in gure 4.
17 2 Structure and Diversity of Gap Junction Channels
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 gure 5. This twisting motion closing the central channel is
possible since the -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].
18 2 Structure and Diversity of Gap Junction Channels
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
-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 dierent isoforms of gap junctional connexins which can be distinguished
with regard to their molecular weight. These dierences are mainly due to
various lengths of the C-terminal loop.
The smallest connexin is Cx26 with an approximate molecular weight of
2126 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 gure 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 gure 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.,
19 2 Structure and Diversity of Gap Junction Channels
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 gure 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 g. 8).
The rst 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 eective 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 gure 7. Cx37 is an isoform belonging to the same branch of the Cx family
20 2 Structure and Diversity of Gap Junction Channels
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 endothe-
lium[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 gure 7.
21 2 Structure and Diversity of Gap Junction Channels
Table 1.
Residue Human Rat Xenopus
124 D E D
16 I V V
234 K R K
251 S T N
253 A P A
257 A S G
263 Q P P
344 S A M
1
347 L V G
1
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 broblasts [Musil et al.,
1990a], in astrocytes and leptomeningeal cells [Dermietzel et al., 1989; Yama-
moto 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 gure 7.
Using a rat Cx43 probe and a 10-day chick embryo cDNA library, chick
Cx42 (see above) and chick Cx45 were identied [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 gure 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
22 2 Structure and Diversity of Gap Junction Channels
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 identied 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 dierences 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 dierences 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
23 2 Structure and Diversity of Gap Junction Channels
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 (g. 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 (gures 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.
24 2 Structure and Diversity of Gap Junction Channels
3
...........................
Distribution of Gap Junctions in the Heart
In the rst 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 bers 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 bers to the ventricular myocytes. Thus, the coupling
within the tissue and between various cells becomes an important feature to
provide the normal impulse conduction. Fromthe above-mentioned considera-
tions an association of the dierent functions and demands with dierent
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. Briey,
to freeze-fracture cardiac muscle specimens, these specimens have to be xed
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 speci-
mens have to be unidirectionally stained in vacuo by exposure to platinum
25
Fig. 9. Distribution of the gap junctions, desmosomes and fascia adherens in an intercal-
ated 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 45, followed by deposition of carbon at an
angle of 90. Details of the method and variations in this technique are de-
scribed 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 intercal-
ated 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 nger-like processes and the interface between
the myocytes parallel to the ber axis as shown in gure 9.
Fromgure 9 it becomes clear that the fascia adherens is located transverse
to the ber 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
26 3 Distribution of Gap Junctions in the Heart
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
dierence because the electrical transfer from one cell to another will be
inuenced by this structural arrangement. This would aect the spatial distri-
bution of current owing from one cell to another and could possibly aect
the ecacy 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 en-
abled 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 m/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 10
3
channels are located in 1 m
2
gap
junction (rat right ventricular myocardium) [Chen et al., 1989].
The surface of the intercalated disk is occupied to 5.70.6% by gap
junctions (canine atrium) [Spira, 1971], 3.3% (right bundle, calf ) [Arluk and
Rhodin, 1974], or even 12.715.1% 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 prole length has been estimated to be in the order of
3.23.8 m/100 m intercalated disk length with 1112 gap junctions/100 m
intercalated disk length and a mean gap junction prole length of about 0.3 m
[Satz 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 Cx43-
positive 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 [Haeiger 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 specic 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
27 3 Distribution of Gap Junctions in the Heart
Fig. 10. Photomicrograph demonstrating immunohistochemical staining for Cx43 in
cryostat sections of the left ventricle of the rat heart. Objective: 40. neouor achroplan,
Zeiss. Magnication: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 nd
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--smooth muscle actin antibody which
is known to specically stain sinoatrial node cells. Thus, it might be possible
that the Cx43-positive cells were obtained fromthe 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.
28 3 Distribution of Gap Junctions in the Heart
Cx40 was found in sinus node cells, atrium, AV node, AV bundle and
bundle branches and Purkinje bers. Cx45 was expressed at low levels in
Purkinje bers 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 com-
posed 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 fromthe 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 -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 fromthe hyperpolarizing inuence of
the atrium (which has a more negative resting membrane potential than the
node) by gradually coupling due to geometric factors (interdigitating bers)
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 immunohisto-
chemically [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 [Satz
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 hypothe-
sized that in the AV node other gap junction proteins may occur than in the
ventricular myocardium. Indeed, only low expression of Cx43 has been ob-
served in rat AV-nodal tissue [Gourdie et al., 1992; van Kempen et al., 1991].
In accordance with this nding, Oosthoek et al. [1993a] could not detect Cx43
expression in human or bovine AV-nodal tissues. However, besides Cx40 Davis
29 3 Distribution of Gap Junctions in the Heart
et al. [1995] did nd 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 [Oos-
thoek 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 bers cells are connected via Cx43 and Cx40. A
threefold higher expression of Cx40 mRNA as compared to Cx43 mRNA
occurs in the canine Purkinje bers [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 ndings. A slightly enhanced amount of Cx43
mRNA in Purkinje bers as compared to ventricular muscle has been demon-
strated in the study mentioned above. This dierence in connexin distribution
and density may contribute to the well-known dierences in conduction prop-
erties [Purkinje fiber conduction velocity: up to 23 versus 0.30.4 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 bers of the rat. Gros et al. [1994]
also stated that gap junctions of rat Purkinje bers contain Cx43 and Cx40.
In the human and bovine heart Cx43 is expressed in Purkinje bers. 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 bers 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].
30 3 Distribution of Gap Junctions in the Heart
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 ex-
pressed 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 identied 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 investiga-
tion 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 [Beny 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 identied: junctional plaques were seen
in the human corpus cavernosum and in the rat aorta with diameters of
0.20.5 m [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 gures 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 dierences observed
by dierent investigators.
Gap junctions between endothelial cells in the vascular wall contain chan-
nels 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.
31 3 Distribution of Gap Junctions in the Heart
Fig. 11. Immunohistochemical localization of Cx43 in a rabbit coronary arteriolar
vessel. The lower gure gives the corresponding phase contrast microphotograph.
32 3 Distribution of Gap Junctions in the Heart
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 interconnec-
tions 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 pro-
cesses of the smooth muscle cells passing through fenestrae of the elastica
interna [Beny and Pacicca, 1994].
In addition, myocytes and broblasts 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 broblasts about 22 pS and between myocytes and
broblasts 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 regard-
ing crosstalk between various tissues and developmental phenomena may be
involved.
33 3 Distribution of Gap Junctions in the Heart
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 immunouorescence on disaggregated
canine ventricular myocytes, Kanter et al. [1993c] could demonstrate with
laser scanning confocal microscopy and electron microscopy that cardiac myo-
cyte gap junctions contain multiple channel proteins with Cx40/Cx43 and
Cx43/Cx45 colocalization, i.e. an individual cell contains more than one con-
nexin isoform. There are two principally dierent 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 myo-
cytes 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 86236 pS and a great sensitivity to transjunctional voltage and those made
from Cx43 2867 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 intercal-
ated 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 dier-
ent compartments.
34 3 Distribution of Gap Junctions in the Heart
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 connex-
ins will be described. In the rst part a special focus is on the physiological
regulation of gap junctional opening and closure by calcium, sodium, magne-
sium, cAMP, ATP, pH, pCO
2
, 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, dierences between ndings with double-cell voltage-
clamp 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, in-
tracellular calcium and pH have been proven to be important regulators of
channel function. In this chapter the short-term regulatory processes are con-
sidered, 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 (g
j
) of neonatal rat heart cells varies with
temperature (37 C, 48.3 nS; 14 C, 21.4 nS; 2 C, 17.5 nS) [Bukauskas and
Weingart, 1993] so that g
j
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
dierent tissues or in dierent species. Thus, generalizations should be avoided
and the specic condition has to be taken into account. One of the rst 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 [Saez et al., 1986, 1990]. Similarly, an increase in junctional conduc-
35
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 hepato-
cyte Cx32 has been attributed to a phosphorylation of Ser-233 which is embed-
ded 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 [Saez 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
inuence 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 bers also increased gap junction
coupling [De Mello, 1984] within 6090 s after injection of the nucleotide.
The role of cAMP and ATP is further elucidated by the nding 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 nding 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 inuenced by PKA
activation [Kwak et al., 1995a]. In the same model, transfectants (SKHp1/
Cx26 and SKHep1/Cx43) were investigated demonstrating that PKA did not
inuence conductance of Cx43 or Cx26 channels either. However, the possibil-
ity that PKA alters the open probability of the channels could not be ruled
out in this study since the eects 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 sh 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 identied). Inhibition of phosphodiesterase with IBMXafter stimulation
of adenylate cyclase using forskolin resulted in an increase in intracellular
36 4 Function and Physiology of Gap Junction Channels
Table 2. Synopsis of the inuence of dierent protein kinases on cell-to-cell-coupling
Protein kinase Connexin Tissue Permeability g
j
Reference
PKA 43 Rat cardiac myocytes 0 0 Kwak and Jongsma [1996]
43 Cardiac myocytes ! ! DeMello [1988; 1991]
Burt and Spray [1988a]
32/26 Rat hepatocytes ! Saez et al. [1986, 1990]
43 Myometrium Cole and Gareld [1986]
43 Sertoli cells Grassi et al. [1986]
45 SKHep1 cells 0 0 Kwak et al. [1995a]
43 SKHep1/Cx43 cells 0 0 Kwak et al. [1995a]
26 SKHep1/Cx26 cells 0 0 Kwak et al. [1995a]
PKC 43 Rat cardiac myocytes ! Kwak & Jongsma [1996]
43 Cardiac myocytes ! Spray and Burt [1990]
43 Rat cardiac myocytes Mu nster and Weingart [1993]
43 Rat cardiac myocytes Doble et al. [1996]
43 Sertoli cells ! Grassi et al. [1986]
? Rat epidermal cells Gainer and Murray [1985]
45 SKHep1 cells 0 !1 Kwak et al. [1995a]
43 SKHep1/Cx43 cells !3,4 Kwak et al. [1995a]
26 SKHep1/Cx26 cells 5 Kwak et al. [1995a]
PKG 43 Rat cardiac myocytes Kwak and Jongsma [1996]
43 Cardiac myocytes Burt and Spray [1988a]
45 SKHep1 cells 0 0 Kwak et al. [1995a]
43 SKHep1/Cx43 2 Kwak et al. [1995a]
26 SKHep/Cx26 0 0 Kwak et al. [1995a]
Tyr-kinase 43 Mouse broblasts Crow et al. [1990]
0>No eect; permeability > as assessed by dye coupling; g
j
>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 conduc-
tance reduced; 5>frequency of 140150 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 unaected, but that the reduced overall conductance can be
ascribed to a decreased open probability [McMahon et al., 1989].
In addition to the eect of cAMP or PKA activation alone, dierent 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 Ca
2+
, injection of cAMP resulted in a biphasic change
37 4 Function and Physiology of Gap Junction Channels
in gap junction conductance with rst an increase followed by a decline in g
j
which was reversible on application of EGTA [De Mello, 1986a].
Besides this, long-term eects of PKA activation and cAMP are known
and are described in the following chapter. It is possible that some of the
contradictory ndings reported in the literature are due to species or tissue
dierences, or to dierences in the intra- and extracellular calcium concentra-
tions or to dierent time courses, i.e. if eects after a longer period are observed
(let us assume longer than 15 min), besides direct inuence on gap junction
conductance, an involvement in protein synthesis can also contribute to the
total eect. In such cases single-channel measurements can be of great advan-
tage.
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 eect of membrane-receptor activation. Receptors being linked to the PLC
system include, among others, the adrenergic
1
receptor, the histamine H
1
receptor and the vasopressin V
1
receptor. However, it should be kept in mind
that noradrenaline, histamine and vasopressin can also activate adenylate
cyclase via |, H
2
or V
2
receptors, respectively. Thus, the eects seen with any
of these mediators and probably with many other mediators can be a composite
eect. In addition, while inositol triphosphate mediates a transient elevation
of intracellular calcium, PKC phosphorylates seryl and threonyl residues at
specic sites [Berridge, 1984]. Thus, receptor activation of PLC-linked receptors
normally causes a double eect: an increase in intracellular calcium and PKC-
dependent phosphorylations, both of which can aect gap junction channels.
To study the eects 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 4-phorbol 12,13-didecanoate. How-
ever, a number of isoforms of PKC exist in cardiac tissue including PKC,
PKC|, PKCc, PKC and PKC (rabbit heart) [Rouet-Benizeb et al., 1996].
Interestingly, only PKC was found to be located close to the intercalated
disks in this study. TPA treatment is assumed to result in a rapid translocation
of PKC and PKCc 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 dierences in the subtypes of PKC
responsible for the eect may contribute to some of the dierences observed
between various preparations.
Very early in gap junction research an eect 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,
38 4 Function and Physiology of Gap Junction Channels
1979]. A similar result was obtained using a DAG analogue, 1-oleoyl-2-acetyl-
sn-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 cAMP-
dependent protein kinase, PKC, or Ca
2+
/calmodulin-dependent protein kinase
II [Saez et al., 1990; Traub et al., 1989; Zhang and Nicholson, 1989], whereas
Cx32 [Saez 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 g
j
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 double-
cell voltage-clamp method, but decreases permeability as assessed by dye coup-
ling in neonatal rat cardiomyocyte gap junction channels. Thus, permeability
for small molecules and electrical conductance do not seemto 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 eect of PKC, PKAand 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 trans-
fering molecules as lucifer yellow from one cell to another under control condi-
tions, was transfected with Cx43 or Cx26. The absence of dye transfer in cells
only expressing Cx45 was not inuenced by 8-Br-cAMP(PKAactivation), 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 trans-
fectant 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 (to-
gether with the 22- and 36-pS conductances observed before). Thus, Cx43 may
be regulatedposttranscriptionally viaPKC-dependent phosphorylation. Onthe
other hand, the inuence of PKC on Cx26 channels is expected to depend on
another mechanism, yet unknown, since Cx26 is not phosphorylated according
to the ndings of Traub et al. [1989] and Saez 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
39 4 Function and Physiology of Gap Junction Channels
as 8-Br-cGMP or |-phenyl-1,N
2
-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 eect 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
broblasts 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 cardiomyo-
cytes 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 6070 and 90100 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 ndings the protein kinase
inhibitor, staurosporine (i.e. preventing phosphorylation), induced a higher
occurrence of the 100 pS events. These ndings indicate that phosphorylation
goes alongwiththeincreasedoccurrence of 60- to70 pSevents anddephosphory-
lation with 100 pS events [Moreno et al., 1992, 1994b].
Several ions are involved in the regulation of gap junctional conductance
including Ca
2+
, Mg
2+
, H
+
and Na
+
. Very early Ca
2+
-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
uorescent dye, aequorin, Rose and Loewenstein [1975, 1976] demonstrated
parallel changes in pCa and uncoupling, concluding that the actual pCa
i
is
40 4 Function and Physiology of Gap Junction Channels
responsible for uncoupling. However, according to Noma and Tsuboi [1987]
intracellular calcium concentrations exceeding about 1 mol/l, i.e. pCa lower
than 6, are needed to aect 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 coecient
remaining constant at about 3.4. Lowering the pH shifted the g
j
-Ca
2+
relation-
ship to the right, i.e. higher calcium concentrations were required for half
maximal depression of g
j
. Noma and Tsuboi [1987] concluded from these
experiments that Ca
2+
and proton compete for negatively charged binding
sites at the Ca
2+
-receptor site involved in the control of g
j
. They hypothesized
that the negative charges necessary for calcium binding may be neutralized
by the protons and proposed a cooperative receptor model:
H
2n
R ACCBR ACCBCa
n
R,
2n H n Ca
with R>Ca receptor and n>number of Ca-binding sites per receptor, dedu-
cing from this the normalized junctional conductance g
j
to follow the equation:
g
j
>1(1/{1+(K
Ca
/[Ca])
n
(1+ ([H]/K
H
)
2n
)})
with K
Ca
being the [Ca
2+
] required for half-maximal depression of g
j
and K
H
the [H
+
] necessary to induce 50% protonation of the receptor. The apparent
half-maximum Ca
2+
concentration (K
E
Ca
) obtained experimentally is described
by the equation:
(K
E
Ca
)
n
>{K
n
Ca
(1+([H] /K
H
)
2n
)}.
With the assumption of n>3 Noma and Tsuboi [1987] calculated K
Ca
and K
H
to be 3.16 10
7
and 1.12 10
7
mol/l, respectively. Since on the other hand the
pH-g
j
relationship was not inuenced by Ca
2+
, 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 aect g
j
in adult heart cells [Ru disu li and Weingart, 1989, 1991], but higher
changes in [Ca
2+
]
i
reduce g
j
in guinea-pig and rat hearts [Maurer and Weingart,
1987]. Maurer and Weingart [1987] concluded from their experiments that re-
duction in g
j
occurs if the intracellular calcium concentration exceeds the
range of 320560 nmol/l, which is below the value proposed by Noma and
Tsuboi [1987]. Maurer and Weingart [1987] argued that the dierence might be
due to dierent stability constants for the calcium buer used to calculate the
cytosolic Ca
2+
concentration. It has beensuggestedthat the bindingsite for Ca
2+
andH
+
is locatedonthe cytoplasmic loopof Cx43[SprayandBurt, 1990]. White
et al. (1990) showed that rises in [Ca
2+
]
i
did not aect g
j
if pH was maintained
at 7.0.
41 4 Function and Physiology of Gap Junction Channels
The 17-kD protein calmodulin acts as an intracellular Ca
2+
receptor
transducing the Ca
2+
signal. A considerable number of the eects are mediated
by the activation of Ca
2+
-calmodulin-dependent protein kinases [Blackshear
et al., 1988; Cheung, 1982]. Since the rst report on a possible involvement
of calmodulin in the regulation of gap junction intercellular communication
[Peracchia et al., 1983] calmodulin-binding sites have been identied 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 specu-
lated that at least in part the calcium eects may be transduced by Ca
2+
calmodulin.
As described, the intracellular pH is an important regulator of g
j
. In-
tracellular acidication is known to decrease junctional electrical coupling in
cardiomyocytes and in Purkinje bers [Burt, 1987; Reber and Weingart, 1982].
g
j
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-g
j
relationship
was principally not aected by intracellular pCa. They found a Hill coecient
of about 2.4, indicating the number of proton-binding sites per receptor and
a half-maximal concentration of 6.1 (pK
H
). In neonatal rat heart cells Firek
and Weingart [1995] found a similar pK
H
with 5.85. One H
+
-binding site could
be identied as histidine-95 in cardiac Cx43 by Ek et al. [1994]. Hermans et al.
[1996] investigated the eects of site-directed mutations in Cx43-transfected
SKHep1 cells by exchange of His-126 and His-142 and found an uncoupling
eect of acidication related to the position of histidines in the cytoplasmic
loop rather than to the total number of histidines. They reported that a fall
in pH
i
caused a reduction in open-channel probability but not in channel
conductance. Using the NH
3
/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 single-
channel 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),
42 4 Function and Physiology of Gap Junction Channels
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 acidication 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 K
H
value on closing the cardiac
gap junction is estimated to be in the order of 7.94 10
7
mol/l, which is dierent
from that describing the eect of H
+
on the g
j
-pCa relationship (1.12 10
7
mol/l;
see above) suggesting dierent binding sites.
In craysh 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 (R
j
) with a concomitant fall in pH
i
, but the
recovery curve for pH
i
was slower than that for R
j
. A concomitant increase
in intracellular [Ca
2+
] was observed so that it was concluded that the pH
i
eect on cellular uncoupling in craysh septate axon is mediated by calcium.
Thus, generalizations of the various mechanisms should be avoided and the
specic experimental model has to be taken into account.
Fromtodays point of viewthese acidication experiments may be contam-
inated by the eects of the pH
i
-regulating pumps, for example the Na
+
/H
+
exchanger and the Na
+
/HCO

3
symport as well as other systems, e.g. the Na
+
/
Ca
2+
exchanger [Doering et al., 1996] and possibly the Na
+
/Ca
2+
-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 load-
ing. In addition, inhibition of proton extrusion with 1 mmol/l amiloride was
reported to enhance the eects of changes in pH on g
j
[Firek and Weingart,
1995].
As a decrease in pH
i
an elevation in pCO
2
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 prepara-
tions. The CO
2
-induced eect is reversible. The decrease in coupling after
exposure to CO
2
has been ascribed to the consecutive fall in pH
i
[Kolb and
Somogyi, 1991]. An eect of increasing CO
2
in the ventilation air of anesthe-
tized 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 pCO
2
is seldom, and is often (in pathophysiological situa-
tions for e.g. myocardial infarction) accompanied by other changes includ-
ing depolarization, potassium eux and many others, which will also aect
the sodium channel availability, which will reduce longitudinal propaga-
tion velocity. That means that, under pathophysiological conditions, aec-
43 4 Function and Physiology of Gap Junction Channels
tion of the gap junctional channel will probably not be mono- but multi-
causal.
Increasing Mg
2+
has also been reported to cause a fall in junctional
conductance in pairs of adult guinea-pig cardiomyocytes [Noma and Tsuboi,
1987]. This eect 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:
g
j
>1(1/{1+(K
E
Mg
/[Mg])
n
}),
with K
E
Mg
>3.16 10
3
mol/l (pK
E
Mg
>2.5) and n>3. A Hill coecient of 3.0 was
calculated. Since the slope of the pCa-g
j
and the pMg-g
j
relationships were
similar, it was suggested that both divalent cations bind to the same receptor
site. The uncoupling eect 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 Mg
2+
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 g
j
/g
j 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
+
/Ca
2+
-
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 bers as indicated by an increase in
input resistance. It is uncertain whether this was a direct eect of sodium or
may be secondary to a rise in intracellular calcium via the Na
+
/Ca
2+
-exchange
mechanism.
As described in the paragraphs above, conclusions on a direct eect of
any of these ions should be drawn with caution, since a change in the concentra-
tion 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
+
/Ca
2+
-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
R
j
in cow ventricular trabeculae indicating that the intracellular ATP content
44 4 Function and Physiology of Gap Junction Channels
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 g
j
[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 deter-
mined the Hill coecient for ATP with 2.6 and the half maximum cytosolic
ATP concentration in the order of 0.68 mmol/l, suggesting a total uncoupling
(g
j
>0) if ATP is reduced below 0.1 mmol/l. The decrease in g
j
induced by
lowering [ATP] to 0.5 mmol/l was not reversible by adding ADP (10 mmol/l)
or 50 mol/l cAMP or 1 mol/l of the catalytic subunit of cAMP-dependent
protein kinase in these experiments. Thus, at Ca
2+
, Mg
2+
and H
+
concentra-
tions considered to be approximately in the physiological range, [ATP] acts
as a regulator of g
j
independent of cAMP-dependent phosphorylation. The
authors suggested a direct eect of ATP via a specic ligand-receptor inter-
action with the gap junctional proteins. Using metabolic inhibition by 2,4-
dinitrophenol (or decrease to 0.1 mmol/l ATP) in adult guinea-pig cardiomy-
ocytes Morley et al. [1992] also described an increase in gap junction resistance
6 min after addition of 2,4-dinitrophenol. However, the increase in R
j
was too
small to impair cell-to-cell propagation in this experimental system.
With regard to other possible regulators of g
j
, arachidonic acid, which
can be released from membrane phospholipids by activation of phospholipase
A
2
secondary to activation of a variety of receptors, has been investigated.
It became obvious from these experiments that in very high concentrations
(50100 mol/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 eect, 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 eect 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 investi-
gated by Massey et al. [1992], who described a concentration-dependent eect
of arachidonic acid on g
j
in neonatal rat heart cells in a physiologically more
relevant concentration range from 2 to 20 mol/l. This uncoupling eect could
be antagonized by inhibition of lipoxygenase with U70344A, but not with
indomethacine suggesting that the arachidonic acid eect at that concentration
is mediated by a lipoxygenase metabolite, e.g. a leukotriene, but not by a
45 4 Function and Physiology of Gap Junction Channels
cyclooxygenase metabolite. The incorporation of arachidonic acid in that
study was 0.695 mol min
1
. Thus, at least two eects of arachidonic acid have
to be considered: (1) an unspecic eect 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 cardio-
vascular system. Thus, it may also play a role in the control of intercellular
communication. Very early in gap junction research the eect of acetylcholine
as an important transmitter on gap junction conductance has been investigated.
First, Petersen and Ueda [1976] demonstrated an increase in junctional resis-
tance in pancreatic acinar cells following the application of acetylcholine.
Concomitantly, the release of amylase was stimulated. A minimum concentra-
tion of 1 mol/l acetycholine was required to evoke uncoupling. The next
question was, how is the acetylcholine eect 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 eect in a double whole-cell technique
although calcium was strongly buered using 20 mmol/l EGTA in the pipette
solution. PKCstimulation has been discussed to participate in the transduction
of the acetylcholine eect [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 nally
22.7% after application of 10
6
and 10
4
mol/l acetylcholine, respectively. The
eect occurred within 10 min. A similar result was found in rat pancreatic
acinar cells following the administration of 5 mol/l acetylcholine, which re-
sulted in cellular uncoupling (dye coupling method) and a 4- to 5-fold increase
in amylase release [Chanson and Meda, 1993]. This eect was independent
of cycloxygenase, calcium and PKC, but could be inhibited by 1 mol/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
eect of acetylcholine. They reported a reduction in the intercellular current
(I
j
) in response to 100 mol/l of the parasympathomimetic drug, carbachol,
which could be mimicked by 8-Br-cGMP. The eect only occurred in the
whole cell patch conguration, but not in the perforated patch conguration,
suggesting that a cytosolic enzyme is necessary for the eect which is washed
out by the pipette in the whole cell patch. Since they found the carbachol
eect 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.
46 4 Function and Physiology of Gap Junction Channels
Fig. 13. Synopsis of the physiological regulators of gap junction channels. >PKC
changes the substate of conductance, *>not found by all investigators as dierences 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, adren-
aline and noradrenaline. In acinar submandibular gland cells of the rat the ad-
ministration of 10
4
mol/l adrenaline elicits a reduction in dye coupling from 97
to75.3%dye-coupledcells [Kannoet al., 1993]. This couldnot be mimickedwith
isoprenaline, but was inhibited with phenoxybenzamine. Thus, the uncoupling
eect of adrenaline in this preparation is mediated by stimulation of the -adre-
noceptor, whereas a stimulation of the |-adrenoceptor has no eect.
In contrast, stimulation of the |-adrenoceptor in the heart increases inter-
cellular 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
eect will occur which is dicult 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 ber axis,
will give insight into the global eect of such a manipulation. De Mello [1986b]
reported that adrenaline increased the spread of electrotonic potentials during
47 4 Function and Physiology of Gap Junction Channels
diastolic depolarization in canine Purkinje bers probably due to a rise in
intracellular [cAMP].
Very recently an eect of the broblast 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 incuba-
tion 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 261270. 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 ndings, FGF-2 induced an increase in Cx43
accumulation and, as a result, an enhancement of coupling between cardiac
broblasts and capillary endothelial cells, respectively [Doble and Kardami,
1995; Pepper and Meda, 1992].
The mechanisms described so far are synoptically summarized in gure 13.
An important point to mention is that, as already said, the aection of the
gap junction conductance is not mono- but multicausal under the most physio-
logical and pathophysiological conditions due to the interactions between the
intracellular mediators. Thus, most processes will aect intracellular calcium
and, on the other hand, a change in intracellular calcium will activate a variety
of intracellular mechanisms and aect the activity of many calcium-dependent
enzymes.
4.2 Functions in Heart and Vasculature
What arethefunctions of intercellular communicationchannels intheintact
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 prob-
ably only at the beginning of an understanding of the role of intercellular com-
munication. 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.
48 4 Function and Physiology of Gap Junction Channels
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 junc-
tional communication as well as synchronization of electrical activation. The
electrical coupling between cardiomyocytes mitigates dierences in the mem-
brane potential between these cells, for example in the course of an action
potential if both cells repolarize at dierent timepoints. This results in smaller
dierences 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, sucient gap junctional
communication can be considered as an endogenous arrhythmia-preventing
mechanism. For a detailed discussion of the role of gap junctional communica-
tion in the biophysics of cardiac activation as related to anisotropy, nonuni-
formity 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 inuence 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 dierences 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 Ca
2+
. 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, prosta-
cyclin and nitric oxide can only aect the local vasotone (at the site of release)
or regulate downstream constriction or dilation. Besides this, upstream regula-
49 4 Function and Physiology of Gap Junction Channels
tion has been observed, but not well understood. Often this was ascribed to
the inuence of the perivascular nerves. However, since Beny 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 vasoac-
tivity 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 inuenced or provided by gap junctional coupling
(g. 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 dierent 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 dierentiate between channels exhibiting sensi-
tivity 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
dierence (by clamping the two cells to dierent potentials) is applied for a
short time to a pair of coupled cells and the current necessary for maintenance
of the voltage dierence is measured. In order to achieve such an experimental
50 4 Function and Physiology of Gap Junction Channels
Fig. 14. Experimental setup for double whole-cell patch measurement of the gap junction
current.
setup two voltage-clamp ampliers are connected via a patch-clamp pipette
to either cell (g. 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 ow across the cell membrane and across the junction between both
cells, the current in cell 1 I
1
can be described under these conditions by the
equation:
I
1
>(V
1
/r
m1
)+[(V
1
V
2
)/r
j
]
and accordingly the current in cell 2 by
I
2
>(V
2
/r
m2
)+[(V
2
V
1
)/r
j
],
with V
1
and V
2
being the voltage relative to the holding potential V
H
(e.g.
40 mVin order to inactivate the sodiumcurrent) and r
m1
and r
m2
the membrane
resistance in cell 1 and 2, respectively. If cell 2 is kept at V
H
, currents I
1
and
I
2
can be described as:
I
1
>(V
1
/r
m1
)+(V
1
/r
j
)
I
2
>V
1
/r
j
51 4 Function and Physiology of Gap Junction Channels
so that I
2
is a direct measure of the current owing 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 dierent resistances (for more details see chapter 8). The
current measured is then plotted against the transcellular voltage V
j
. Linear
regression reveals the total intercellular resistance. g
j
follows the equation:
g
j
>N
j
P
0
,
with N>number of channels capable of opening and closing, P
0
being the open
probability and
j
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 ampliers
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 I
j
versus V
j
may reveal a linear relation as
shown by Veenstra et al. [1993], which means that the instantaneous gap
junctional conductance is constant, i.e. g
j
>I
j
/V
j
>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 rectication is present. Rectication 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 tting the normalized g
ss
/V
j
relationships with the
two state Boltzmann distribution which follows the function:
g
ss
/g
inst
>{(g
max
g
min
)/(1+exp[A(V
j
V
0
)])}+g
min
according to Spray et al. [1981] and Veenstra et al. [1993] with g
max
being the
maximum conductance (>1, normalized to instantaneous g
j
) and g
min
the
minimum conductance. V
0
is the half-inactivation voltage where g
j
is between
g
min
and g
max
and A>zq/kT (z>number of equivalent electron charges, q>
voltage sensor, k>Boltzmann constant, T>temperature).
52 4 Function and Physiology of Gap Junction Channels
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 ampliers (SEC05).) For pipette solution,
etc., see chapter 8.
a
b
Using the technique of double whole-cell patch reveals data as shown
in gure 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
53 4 Function and Physiology of Gap Junction Channels
ranging from 50 to +50 mV were applied. From these experiments the I/V
relationship in gure 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 regu-
lated by transcellular voltage or not, the reader may be confused by the various
ndings of dierent 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 rela-
tions have been demonstrated to be linear, i.e. g
j
is independent of transjunc-
tional voltage and rectication 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 communi-
cate by only a few gap junction channels, linear current-voltage relationships
for instantaneous and steady-state g
j
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 (neo-
natal hamster cardiomyocytes) [Veenstra, 1990]. Outside this range rectication
is observed and the slope of g
ss
declines progressively to values near zero [Rook
et al., 1988; Veenstra, 1990, 1991a], whereas the instantaneous g
j
remains
constant. This voltage-dependent behavior of g
ss
can be tted 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
j
but in the ratio t
open
/t
closed
, which is decreased
so that at a given time point more channels are in the closed state.
Spray et al. [1985] found g
j
to be unaected by the transjunctional potential
gradient and by the membrane potential in dispersed and reaggregated rat
ventricle cells. Similarly, Kameyama [1983] reported that R
j
is independent of
the transjunctional potential gradient. While the instantaneous g
j
is insensitive
to the transjunctional voltage, in most cases g
ss
can exhibit voltage dependence
in some preparations [Veenstra et al., 1993] (g. 16). Voltage dependence in
neonatal cardiac myocytes can especially be observed with large transjunc-
tional voltages in the order of 80 mV or more.
Rectifying behavior was observed in craysh axons with depolarization
at the presynaptic side increasing the junctional conductance [Furshpan and
Potter, 1959; Giaume et al., 1987] and in sh [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.
54 4 Function and Physiology of Gap Junction Channels
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].
a
b
Do all connexins exhibit the same sensitivity to the transjunctional volt-
age? Trying to answer this question, Nicholson et al. [1993] have shown in an
xenopus oocyte expression system that g
ss
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 g
ss
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 V
j
,
respectively, indicating a sensitivity of g
ss
of the Cx40 channel to transjunctional
55 4 Function and Physiology of Gap Junction Channels
Table 3. Boltzmann equation parameters for various connexins
Connexin G
min
V
0
[mV] Slope factor References
37 0.27 28 0.08 Reed et al. [1993]
40 0.33/0.28 54/+47 0.13/0.11 Beblo et al. [1995]
0.32 35 0.225 Ebihara [1995]
43 0.37
a
60 0.106 Moreno et al. [1995]
45 0.072 13.4 0.115 Moreno et al. [1995]
0.17/0.16 16/+22 0.126/0.115 Ebihara [1995]
If two values are given, the data on the left are mean values at negative V
j
and on the
right mean values at positive V
j
. It becomes evident that Cx43 exhibits the lowest sensitivity
to V
j
since the half inactivation voltage V
0
is highest, whereas according to these data Cx45
and Cx37 exhibit the strongest V
j
dependence.
a
G
min
/G
max
.
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 V
0
45/49 mV and g
min
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, depend-
ing on the pipette solution used, V
0
51/51 mV, g
min
0.28/0.25 and z 3.1/2.9
(KCl solution), or V
0
59/59 mV, g
min
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 V
0
73.7/65.1 mV and g
min
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 dierent 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 V
0
60.5/59.5 mV, g
min
0.27/0.29.
The problems arising from the various ndings regarding the voltage
sensitivity of g
ss
initiated a very elegant study by Jongsma et al. [1993] answering
the question Are cardiac gap junction channels voltage sensitive?. In a com-
puter simulation they modelled two cardiomyocytes interconnected via a gap
junction and varied the number of gap junction channels within this intercon-
56 4 Function and Physiology of Gap Junction Channels
nection. The open probability of a single channel was assumed to follow the
equation:
p
0
>1/{1+exp([A

+A
|
][oV
j
V
0
])},
with > exp(A

[oV
j
V
0
]) and |> exp(A
|
[oV
j
V
0
]) and A

(>0.041 mV
1
) and A
|
(>0.021 mV
1
) being the voltage sensitivities of and |,
(>2.5 s
1
), the rate at which equals | and V
0
(>74 mV), the transjunctional
voltageat whichequals |(thedatawere obtainedfromsingle-channel measure-
ments carried out on neonatal rat heart cells by Rook et al. [1988]). They found
arelationshipbetweenthe pipette series resistance andthe appearance of voltage
insensitivity of g
ss
. It was shown that gap junctions with intermediate numbers
of channels (130or morechannels wereassumed) appear tobevoltageinsensitive
if pipettes with 60 MD are used and gap junctions with 300 or more channels if
20 MDpipettes are used. In real measurements the circuit is even more compli-
catedbythe fact that, part of the junctional current is shuntedtogroundthrough
the membrane resistance. The higher the series resistance the more dicult 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 transjunc-
tional 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 reect a change in the regulation of junctional resis-
tance 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 g
ss
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 inuence 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 inuence. In these cells potential
gradients high enough to disturb for example the pacemaker function may
arise and, thus, such a disturbing inuence may be prevented by transjunctional
voltage-sensitive gating of the gap junctions. This ts with the general nding
that Cx40 and Cx45 are more sensitive to transjunctional voltage than Cx43.
However, there is at present no clear evidence for these hypotheses.
57 4 Function and Physiology of Gap Junction Channels
What is the real range of gap junction resistance measured in various
systems? Kameyama [1983] reported in reaggregated cell pairs of adult guinea-
pig hearts R
j
in the order of 1.42.1 MD (corresponding to 476714 nS) and
Noma and Tsuboi [1987] 0.2511 MD(903.900 nS) in adult guinea-pig cardio-
myocytes with a peak in the distribution around 1,000 nS (i.e. 1 MD). Weingart
[1986] calculated the gap junction conductance in adult cardiomyocytes with
204 nS (4.9 MD). In neonatal or embryonic heart cell pairs lower conductances
were reported: 030 nS (?33 MD) in embryonic chick heart cells from 7-day-
old embryos [Veenstra, 1990] and 0.0535 nS (?28 MD) in neonatal rat heart
cells [Burt and Spray, 1988a].
In cable preparations of various species resistance has been reported in
the order of 200600 Dcm: 523 Dcm in bullfrog trabeculae [Haas et al., 1983];
200250 Dcm in guinea-pig trabecular muscle [Daut, 1982]; 350530 Dcm in
rabbit Purkinje bers [Colatsky and Tsien, 1979] and 588 Dcm in frog ventricu-
lar 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] pre-
dicted the single-channel conductance. According to their considerations and
to Hille [1992], R
Channel
is given by the equation:
R
Channel
>R
Pore
+R
Access
with
R
Pore
>(1t(d/2)
2
)
and
R
Access
>2/td.
Assuming the inner pore to be lled with a 130-mmol/l salt solution equals
100 Dcm and R
Channel
equals 1020 GD. 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;
58 4 Function and Physiology of Gap Junction Channels
Rook et al., 1988] that the regulation of gap junction conductance is not the
all or nothing and not quantal, but graduate so that dierent single-channel
conductances or substates have been postulated [Page, 1991]. This is probably
reected 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 non-
junctional membrane potential [Ru disu li and Weingart, 1989]. In contrast in
the neonatal rat heart Burt and Spray [1988a] found a single-channel conduc-
tance of about 60 pS. The voltage-insensitive component of g
j
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 dened two substates of this channel,
i.e. 6070 pS and a higher conductance state with 90100 pS.
Kwak et al. [1995b] also characterized two substates in neonatal rat cardio-
myocytes with 20 and 4045 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, 4045 and 70 pS. Similarly, in Cx43-transfected SKHep1 cells they found
three dierent substates: 30.59.1, 61.29.8 and 89.112 pS [Kwak et al.,
1995a]. From these dierent ndings one might conclude that at least three
dierent substates of single-channel conductance of Cx43 channels can be
distinguished. Depending on phosphorylation or dephosphorylation by vari-
ous protein kinases the dierent 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
j
(main state) was determined 96 pS (KCl), 61 pS
(Cs-aspartate) and 19 pS (TEA-aspartate) and
j
(residual state) was deter-
mined 23, 12 and 3 pS, respectively, revealing
j
(main state)-
j
(residual state),
ratios of 4.2, 5.1 and 6.3, indicating that the residual state restricts ion move-
ment more eciently 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 (1565 ms). The dier-
ences in the results of various investigators may perhaps be due to dierent
experimental models and conditions. An example of single-channel recordings
is given in gure 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
59 4 Function and Physiology of Gap Junction Channels
Fig. 17. Single-channel conductance of neonatal rat heart cells. Note the residual con-
ductance [Valiunas et al., 1997]. The V
j
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 (12 ms) in contrast to the
transitions between open and closed states (1545 ms).
The third channel found in heart muscle is the Cx45 channel. This was
reported to exhibit an overall conductance of 3.10.4 nS in cell pairs [Kwak
et al., 1995a] with a mean single-channel conductance of 36.56.5 pS or
1.3 nS in human Cx45-transfected SKHep1 cells [Moreno et al., 1995] and
j
of 328 pS. At higher transjunctional voltages an additional conductance
state with 22.54.3 pS was observed. This channel strongly depends on trans-
junctional voltage [Moreno et al., 1995] comparable to Cx38 channels. How-
ever, single-channel conductance is not a function of transcellular voltage.
Besides Cx40, Cx43 and Cx45, Cx37 channels are expressed in the cardio-
vascular 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 conduct-
ance states [Reed et al., 1993]. Several single-channel conductances were found:
60 4 Function and Physiology of Gap Junction Channels
21922, 1656, 12313 and 531 pS at V
j
>3040 mV. At higher V
j
the
large conductance was no longer observed and
j
of 944, 695 and
3910 pS were detected at V
j
>8090 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 macro-
scopic channel conductance is then either inuenced 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 rst important dierence 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 dierence 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: tetra-
ethylammonium (TEA), 4-aminopyridine (4-AP), Zn
2+
, Co
2+
and Ni
2+
. 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 2,7-
dichlorouorescein and to the more polar 6-carboxyuorescein dye. Interes-
tingly, the 2,7-dichlorouorescein permeability did not increase with increas-
ing junctional conductance in that study.
With regard to small molecules other than ions Tsien and Weingart [1976]
reported that
3
H-cAMP diuses across gap junctions in calf and cow ventricle.
Furthermore, Weingart [1974] has shown that
14
C-TEA (molecular weight 130)
diuses transjunctionally in sheep ventricular muscle and he found the channel
61 4 Function and Physiology of Gap Junction Channels
diameter to be in the order of 10 A

. A number of dierent dyes with molecular

weights ranging up to 859 Daltons have been demonstrated to diuse across
the gap junctions including procion yellow (MW 697) in sheep and calf
Purkinje bers [Imanaga, 1974], 6-carboxyuorescein (MW 670; diusion
coecient 5.8 10
6
cm
2
/s), lucifer yellow (MW 457; diusion coecient
3.0 10
6
cm
2
/s), lissamine rhodamine B200 (MW: 859; diusion coecient
8.6 10
7
cm
2
/s), while Chicago blue (MW 1,000 Daltons) did not diuse across
the channels [Imanaga, 1974, 1987; Imanaga et al., 1987]. From these data
the authors calculated an upper limit for the channel diameter of 1.21.3 nm.
In more physical terms the permeability P
j
through a gap junction channel
can be described using the equation:
P
j
>V
cell
k
i
/A
j
,
with A
j
being the area of gap junctional membrane, V
cell
the cell volume and
k
i
the experimentally measured rate constant for transcellular diusion.
Thus, the gap junction channel behaves like a gated pore exhibiting some
selectivity for cations over anions, and acting as a diusion barrier for mole-
cules 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 G
j
is concerned.
The steady-state conductance G
ss
can exhibit a more or less stronger sensitivity
to V
j
depending on the connexin the channel consists of.
62 4 Function and Physiology of Gap Junction Channels
5
...........................
Regulation of Gap Junction Expression,
Synthesis and Assembly
We have considered the structure, diversity and the function of gap junc-
tional 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 assem-
bly. 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 t each other forming the intercellular pore? Research in this area
is still at its beginning and many of the processes involved are not well under-
stood. 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.
63
It has been demonstrated that cell coupling is established within 330 min
after bringing disaggregated cells into contact. Interestingly, the resulting in-
crease 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 oversimplication. 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 diuse 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 rst opening occurred within 725 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 ribo-
somes 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 nal 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 chaperon-
like 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 nal structure [for review see, Hartl, 1996].
The binding of this putative assisting factor was suggested to occur at the
NH
2
terminus of the gap junction protein anchoring the NH
2
terminus to the
cytoplasmic site of the ERmembrane. 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
64 5 Regulation of Gap Junction Expression, Synthesis and Assembly
form was the phosphorylated state of the nascent 40-kD form of Cx43 sug-
gesting 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 phosphory-
lation 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 specic 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 signicant inuence on gap
junction assembly, since in disaggregated cells the formationof intact intercellu-
lar channels can be inhibited by Fab fragments of N-cadherin-specic antibod-
ies. If cadherins are involved, a calcium dependence of the gap junction
assembly process should be detectable. In Noviko hepatoma cells expressing
Cx43, Miner et al. [1995] investigated the sensitivity of the gap junction forma-
tion on extracellular calcium by means of electron microscopy and a dye-
transfer 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 ndings 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 nding 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 car-
diac 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 ndings that such
65 5 Regulation of Gap Junction Expression, Synthesis and Assembly
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 conned 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 rst 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
immunouorescence. 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-eective.
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 gure 18.
66 5 Regulation of Gap Junction Expression, Synthesis and Assembly
Fig. 18. Synthesis, posttranslational modication and assembly of gap junctions.
Structurally it has been found that large gap junctions are surrounded by
small gap junctions (0.5 m in diameter containing 12100 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 conrmed by Severs et al. [1989],
but over a period of 1522 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
67 5 Regulation of Gap Junction Expression, Synthesis and Assembly
well-known run-down of intercellular coupling in the course of such experi-
ments, 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 o and a double-wall vesicle is formed, which is nally
degraded within a lysosome. Within a cell so-called annular gap junctions
have been seen representing circular proles 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 proles and between the layers of the basal lamina covering
the nonjunctional membrane close to the interplicate segment. Negatively
staining with La(NO
3
)
3
revealed that these GJAVs contained laminar structures
which have been identied 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 repre-
sent extracellular tracks for myocyte cell processes helping to meet or to retract
fromthe 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 modi-
cation of Cx43 in neonatal cardiomyocytes. After labelling with
35
S-Met,
immunoprecipitation with anti-Cx43 antibodies followed by SDS-PAGE and
uorography revealed a phosphorylated and a non-phosphorylated form of
Cx43. In pulse-chase experiments the half-life of Cx43 was determined with
12 h, and, furthermore, the turnover rate of the phosphate groups was experi-
mentally dened 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
68 5 Regulation of Gap Junction Expression, Synthesis and Assembly
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 dierent phosphorylation (at vari-
ous sites as in Cx43), may control various aspects of gap junction metabolism
and function.
The ndings of Darrowet 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 272285 and 327340
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, Int 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 cAMP-
response 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]. Saez et al. [1989] reported that cAMP delayed the uncoupling
69 5 Regulation of Gap Junction Expression, Synthesis and Assembly
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 volt-
age-sensitive dyes. However, the molecular mechanisms of this enhanced ex-
pression appeared to be dierent 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 newprotein 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 eect might indicate
the action of a specic promoter or enhancer sequence near the Cx43 gene.
The lack of sensitivity to cycloheximide suggests a possible modication 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 prob-
ably 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 uncoup-
ling eect of phorbol esters if cells are exposed to both agents from the start
of the experiment [Kanno et al., 1984], but this protective eect can be abol-
ished 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
70 5 Regulation of Gap Junction Expression, Synthesis and Assembly
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 Loewensteins
[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 aect Cx43 mRNA or
protein synthesis. With regard to the intracellular distribution of Cx43, FGF-2
exposure decreased immunouorescence-staining intensity at sites of intermy-
ocyte contact and induced phosphorylation of Cx43 in serine and tyrosine
residues. In other cells (cardiac broblasts), however, the same authors demon-
strated 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, 1991ac] isolated the entire gene encoding Cx43 including
about 5,000 base pairs of 5-anking 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
71 5 Regulation of Gap Junction Expression, Synthesis and Assembly
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 5-
anking 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 specicity. These
studies begin to characterize the cis-acting elements of the Cx43 gene. These
regulate the strength and specicity 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 gure 19 according
to De Leon et al. [1994].
Regulation of Cx40 expression has been suggested to be somewhat dier-
ent 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 nd the protein suggesting that either the protein is not translated or after
translation rapidly degraded.
72 5 Regulation of Gap Junction Expression, Synthesis and Assembly
6
...........................
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 arrhyth-
mia. There are many factors involved in the pathophysiology of cardiac isch-
emia 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 [Kleber et al., 1978, Kramer and Corr, 1984] and inux of calcium
which is further enhanced by reduced calcium elimination via Ca
2+
-ATPases
or by exchange of the accumulating sodium against calcium. The mechanisms
of calciumoverload 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 I
K.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
+
-eux. However,
this K
+
-eux 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
bers [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 heteroge-
neous with regard to its local distribution between ischemic and nonischemic
heart. Dierences in wave length are known to cause reentrant arrhythmias
[Allessie et al.,1973; Krinsky, 1981]. Slowing of conduction is assumed to be
73
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 re-
sulting from pump failure) and sympathetic activation with release of nor-
adrenaline 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 long-
chain 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 signicantly reduced the inci-
dence of arrhythmia induced by ischemia in vivo [Corr et al., 1989]. In addition,
Purkinje bers exposed to lysophosphatidylcholines, which may be the case
in subendocardium adjacent to ischemic myocardium, have been shown to
generate early after-depolarizations [Arnsdor and Sawicki, 1981]. Lysophos-
phatidylglycerides in combination with acidosis and elevated [K
+
] can induce
delayed after-depolarizations and triggered activity in isolated Purkinje bers
[Pogwizd et al., 1986].
However, an enhanced extracellular potassiumconcentration and depolar-
ization of the bers 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. Type-
1a arrhythmias occur 210 min after the onset of ischemia with a peak at
56 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 1230 min after the onset
of ischemia with a peak at 1520 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 reviewsee, Janse and Wit, 1989] or are due to gap junctional
74 6 Gap Junctions in Cardiac Disease
uncoupling and disturbed intercellular communication. From the considera-
tions 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 subepicard-
ium) 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 pCO
2
, the sodium
overload of the bers, fatty acids released in the ischemic tissue (see chapter 7),
accumulation of long-chain acylcarnitines, leukotrienes from activated leuko-
cytes, 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 dicult or not possible to ascribe this eect 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
(gure 20).
In a more sophisticated setup Kleber et al. [1987] investigated the eect
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 calciumconcentra-
tion 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-
75 6 Gap Junctions in Cardiac Disease
Fig. 20. Stimulus-response interval in guinea pig papillary muscle under normoxic
and hypoxic conditions. Hypoxia was concomitted with glucose-free superfusion. Note the
signicant increase in the stimulus-response interval after 12 minutes of hypoxia.
tingly, uncoupling occurred concomitantly with the secondary rise in extracel-
lular potassium. This secondary rise was also delayed by inhibition of
acyltransferase I with 10 mol/l 2-(5-(4-chlorophenyl)-pentyl)-oxirane-2-car-
boxylate (POCA). Since it has been shown that long-chain acylcarnitines can
elevate intracellular calcium [Fischbach et al., 1992; Meszaros and Papano,
1990], although inhibiting the L-type calcium current [Wu and Corr, 1992],
Yamada et al. [1994] concluded fromtheir experiments that long-chain acylcar-
nitine-induced uncoupling is due to an eect 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
dicult to ascribe a phenomenon such as cellular uncoupling to only a single
factor. Since other factors occurring in ischemia can also contribute to uncoup-
ling 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
76 6 Gap Junctions in Cardiac Disease
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 cardiomy-
ocytes isolated from rabbit hearts which had previously undergone global
normothermic ischemia followed by 30-min of reperfusion in a Langendor
setup in comparison to cells isolated from hearts which were either perfused
according to the Langendor technique for 75 min (without ischemia) or
isolated directly after removal of the heart without Langendor perfusion. In
all three groups of cells the action potential characteristics were normal. Mean
G
j
was also almost normal in all three groups but was signicantly 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 2030 min of ischemia. Hypoxia lasting for
longer than 30 min induced loss of lipid aisles and in consequence a condensa-
tion of connexons in perfusion-xed rat hearts with a subsequent rapid crystal-
line 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, ultrastruc-
tural changes have been identied. Frank et al. [1987] suggested that rearrange-
ment of sarcolemmal P-faceparticles mayoccur after 5 minandmayresemblean
unspecic response to alterations in membrane uidity accompaning ischemia.
However, the gap junctional surface density, i.e. the gap junction prole lengths
per unit myocytesectional area, is not alteredat theonset of uncouplingat 30 min
of hypoxia, althoughareductionintheP-facecenter-to-center distanceinfreeze-
fracture 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 takeninto account that other elements of the cytoskeletonare alsoimportant
for the assembly of gap junctions as pointed out in the previous chapter and,
thus, shouldalsobe investigatedinthe course of ischemiaandinfarctioninorder
to nd out the primary processes of uncoupling.
What are the consequences of gap junctional uncoupling? Is it a benet
or a risk, or even both? Gap junctional uncoupling on the one hand will lead
77 6 Gap Junctions in Cardiac Disease
to electrical and metabolic isolation of the ischemic tissue. If between this
tissue and the surrounding cells dierences in action potential duration exist,
these will be enhanced since coupling of the cells would smooth these dier-
ences 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
dierences 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 inuence which might induce arrhythmia via
depolarization of Purkinje bers. In addition this uncoupling may provide
some kind of energy-saving eect for the tissue since the ischemic tissue is
no longer activated and will, thus, stop contracting thereby reducing energy
consumption. Thus, both benecial and disadvantageous eects can result
from gap junctional uncoupling. It probably depends on the local spatial
distribution of the electrophysiological changes with regard to the microana-
tomy 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 (1015 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 2060 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 self-
debrillation 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.
78 6 Gap Junctions in Cardiac Disease
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 occur-
rence of cardiac arrhythmia. Changes in conduction properties have been
identied [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 car-
diomyocytes, whereas in broblasts coupling was found to be increased in
response to FGF-2 (Doble and Kardami, 1995]. However, presently it is un-
clear whether this factor aects 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 aect 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 [Satz 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 pre-
dominantly 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 aniso-
tropic conduction and locally reduced conduction velocity forming a pro-
arrhythmic substrate. The active properties of cells and resting membrane
potential can be quite normal in the presence of manifest cardiac arrhythmia,
79 6 Gap Junctions in Cardiac Disease
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 todays research gap junctions are one of the most important
determinants of these passive conduction properties [Peters et al., 1993; Satz
et al., 1992]. In hearts of patients suering 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 fromthe infarct scar) the pattern of gap junction distribu-
tion is disturbed with a wide dispersion of the gap junctions over the whole
cell surface instead of being conned 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 pro-
cesses with gap junctions in the absence of fasciae adherentes. Besides these
changes, annular gap junction proles were found indicating a possible in-
ternalization of gap junctions. Between all these cells normal cells also occur
[Severs, 1994a, b].
Following experimental infarction in the dog heart (10 weeks after occlu-
sion of the left anterior descending coronary artery) [Luke and Satz, 1991],
a more diuse interstitial brosis 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 ecient 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 brotic infarct border zone. Intercalated disk zones were less
clearly dened, 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 Satz, 1991]. They found a reduction in connections of
cells in primarily side-to-side apposition by 75%, while connection of end-to-
end apposed cells were reduced by only 22%. This should result in a dispropor-
tionate increase in resistivity in the transverse direction thus enhancing aniso-
tropy, 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 Kleber, 1993].
80 6 Gap Junctions in Cardiac Disease
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 incorpora-
tion of connective tissue will also alter the pathways of excitation and will
cause inhomogeneity of anisotropy. Besides this, surviving strands of Purkinje
bers 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 m
2
/m
3
)
[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 dierent 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 specic anti-Cx43
antibody for immunouorescence using a laser scanning confocal microscope.
The investigations were carried out with a double-labeling technique using a
second cell-specic antibody. They found a colocalization of Cx43 with smooth
muscle cells but not with macrophages, and conrmed 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 junc-
tions changed and they became more patchy with larger diameters of the
individual junctions.
81 6 Gap Junctions in Cardiac Disease
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 geo-
metry, changes in the -adrenoceptor population, impairment of the regulation
of the intracellular (diastolic) calcium concentration, possibly enhanced endo-
thelin 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 inter-
ested in whether in the course of heart failure gap junctional alterations may
occur.
In patients suering 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 hu-
man left ventricles due to aortic valve stenosis, a general reduction in gap junc-
tion surface area per unit cell volume by about 40% (0.0031 versus 0.0051 m
2
/
m
3
) has beenobserved[Peters et al., 1993]. The gapjunctions inthe 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 ndings, in guinea pigs with cardiac hypertrophy
following renovascular hypertension Peters [1996] reported a substantial in-
crease 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 ndings in hypertrophied human myocardium. However,
the dierent pathophysiology should be taken into account. It might be specu-
lated 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 diusely, 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.
82 6 Gap Junctions in Cardiac Disease
Another important cardiovascular disease aecting 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 dierent 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 ndings 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 un-
coupling, 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 eect
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 eect 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 chronication 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 chronication of arrhyth-
mia 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 brillation 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.
83 6 Gap Junctions in Cardiac Disease
In chronic atrial brillation Van der Velden et al. [1996] recently showed
changes in the Cx40 distribution pattern in goat atria with chronic brillation.
In the goat model used, atrial brillation was induced via chronic high-fre-
quency pacing and they observed that, after switching the stimulus o brilla-
tion 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 brillation. 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 brillation which persisted if stimulation was switched
o. 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 brillation the Cx43
distribution changed with a more disperse pattern without the typical polariza-
tion (gure 21).
Fromthese 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 chronication of the
arrhythmia.
Taken together, all these ndings point to a new understanding of the
arrhythmogenic substrate as a more structural change reecting 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 antiar-
rhythmic 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
84 6 Gap Junctions in Cardiac Disease
Fig. 21. Cx43 immunostaining of rat atria either native (a) or after 24 h of atrial
brillation (b) [Dhein et al., 1997a].
a
b
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
signicantly 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 in-
tracellular Cx43 and only little at the appositional membranes. In addition, the
cellular electrophysiology is altered with shortened action potential, elevated
intracellular resting calciumlevels and altered response to -adrenergic stimuli.
85 6 Gap Junctions in Cardiac Disease
The immunohistochemical ndings 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 specic development. It is now
known that mutations in the gene encoding Cx32 causes X-linked Charcot-
Marie-Tooth disease [Bergoen 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 neo
r
gene which lacked a promotor. Homozygous cell lines were morphologically
normal and dierentiated 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 ospring from
heterozygous crosses was analyzed. No viable homozygous ospring 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 hearts pulmonary outow tract of the
right ventricle. This region was lled with intraventricular septae dividing the
outow tract into separate or blind-ended chambers. Filling of the right vent-
ricle 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 ex-
pressing 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
86 6 Gap Junctions in Cardiac Disease
reported by Britz-Cunningham et al. [1995]. These authors investigated Cx43
DNA from 25 normal subjects and 30 children suering from various congen-
ital heart diseases (including hypoplastic left and right heart syndromes, Di-
George syndrome, septal defects, trisomy 13 and others) using the polymerase
chain reaction. They expressed the mutant DNAin cell culture and investigated
its eect on the regulation of intercellular communication. Within the childrens
group 6 children were identied, all suering from syndromes including com-
plex heart malformations, with substitutions of one or more phosphorylatable
serine or threonine residues. In the Cx43 DNA of 5 of these patients a substitu-
tion 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 ndings
that mutations in the Cx43 gene leading to cell-to-cell communication de-
ciencies 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 conduc-
tion and cardiac contractility. Genome-wide linkage analysis revealed that
polymorphic loci near the centromere region of chromosome 1 (chromosome
1p11q1) were linked to the disease locus with a maximum multipoint lod
score of 13.2 in the interval between D1S305 and D1S176. The authors specu-
lated 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 1pterq12. 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 chro-
mosome 11, it has been sugested that in DDK syndrome the regulation of
intracellular pH is disturbed leading to lower pH
i
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 dierentiation 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.
87 6 Gap Junctions in Cardiac Disease
6.7 Gap Junctions in the Aging Heart
As discussed earlier in this book, it has been shown that there are consider-
able 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 chang-
ing with increasing age as far as senium is concerned. It will be dicult to
elucidate this question because it is necessary to dierentiate 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 microbrosis is observed
which in turn will seperate the bers from each other and thereby enhance
the degree of nonuniformity as discussed in the rst 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 bers. Concomitant changes in the gap junction
distribution are probably secondary to cardiac diseases, although at present
an eect of age per se cannot be excluded.
88 6 Gap Junctions in Cardiac Disease
7
...........................
Pharmacological Interventions at
Gap Junctions
Inthe previous chapters the role of gapjunctions incellular communication
and in cardiac disease has been outlined. It became obvious that in many
diseases the intercellular communication is reduced via gap junctional uncoup-
ling or via reduced or altered expression of gap junctional channels. On this
background a straightforward idea would be to simply enhance the gap junc-
tional coupling by any agent. However, one has to consider that the uncoupling,
as pointed out in the previous chapter, is conned to certain areas, the diseased
areas for example, in the heart. In addition, the uncoupling also has some
positive eects, for example a putative energy-saving eect 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 en-
hanced. Since the channel is also permeable to small molecules it can be
anticipated that molecules like ATP can ow 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
+
, Ca
2+
, K
+
) would
be able to diuse to the nonischemic zone possibly exerting unfavorable eects
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 aected in the way described above. There
might be a similar eect in the close border zone between diseased and normal
tissue, but the eect would be conned to that zone. Inhomogeneities within
the diseased zone would be smoothened whereas the normal tissue behavior
would probably be less aected. This could especially smooth dierences in
action potential duration and thereby prevent, in some situations, from reent-
rant arrhythmia, since this is often related to dierences in action potential
duration, to dispersion. Another important factor in the initiation of reentry
89
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 eect
against arrhythmia if arrhythmia is due to uncoupling. However, if the coupling
eect is unselective, it would probably postpone an impairing eect as discussed
above for ischemia. From this theoretical point of view selective coupling-
enhancing eects 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 eect
during reperfusion due to energy saving. However, a hardly achievable prereq-
uisite would be a selective eect 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 mis-
match between the current source which was represented by strands of cultured
cells of 55 mwidth in their experiments and the current sink being represented
by the expansion of the strand to a rectangular monolayer), failure of antero-
grade activation from the small current source to the large current load oc-
curred, 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 antiar-
rhythmic eects 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 t 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 hep-
tanol and octanol, saturated and unsaturated fatty acids, and alcohols and
90 7 Pharmacological Interventions at Gap Junctions
volatile anesthetics like halothane and ethrane (enurane). 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 eective 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 eect may be
caused by limiting the channels from opening to their largest conguration,
i.e. by interference with the switching between various conductance states
[Chen and DeHaan, 1993]. With regard to the mechanism there is no eect
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 applica-
tion of 3 mmol/l heptanol single-channel behavior can be observed. According
to Ru disu li and Weingart [1989] the eect 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
10
4
mol/l and the maximum eect at a concentration of about 10
3
mol/l
heptanol. K
d
was determined to be 0.16 mmol/l and the Hill coecient z>2.3
for the equation G
j
>(K
d
)
2
/((K
d
)
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 ndings that heptanol uncoupled
cardiac cells via impairment of the open probability p
o
, the gap junction
conductance g
j
being described by the equation g
j
>N
j
p
o
(N>number of
channels). Expression, phosphorylation or localization of Cx43 are not altered
by brief exposure (520 min) to 2 mmol/l heptanol [Kimura et al., 1995]. The
mechanism of action of heptanol was further claried by Bastiaanse et al.
[1993], who showed that the uncoupling eect was based on a decrease in the
uidity 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
+
/Ca
2+
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 eect.
91 7 Pharmacological Interventions at Gap Junctions
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 inuence
of various saturated and unsaturated fatty acids on junctional coupling. They
found saturated fatty alcohols with an acyl chain length of 712 but not higher,
as well as unsaturated C
18
cis 9 fatty alcohol to be eective in uncoupling
neonatal rat heart cells. Saturated fatty acids with acyl groups of 1014 carbons
but not more and unsaturated cis 9 fatty acids with acyl chain lengths of
1418 exhibited an uncoupling eect as well. Decanoic acid in concentrations
of 2 mmol/l rapidly and fully reversibly uncoupled cardiac cells. As with hep-
tanol no change in
j
was observed so that the investigators concluded that
the uncoupling eects were due to a reduction in the open probability rather
than in
j
. These drugs are supposed to incorporate in the lipid bilayer and
to increase disorder in the interior of the membrane (C9C18 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 inter-
action. All drugs listed above found to be eective 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 mem-
brane. 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, de-
canoic acid and palmitoleic acid can uncouple heart cells without aecting
other transmembrane channels contributing to the action potential [Burt et
al., 1991]. Another important feature is the nding that multiple lipophiles
have additive eects [Burt et al., 1993].
Cells, however, dier with regard to their sensitivity to these lipophilic
compounds. Adult rat heart cells, for example, are relatively resistant to un-
coupling 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 dier
in their cholesterol-rich domains. In addition, at present it is not clear whether
there are dierences in the sensitivity of various connexins. Oleic acid for
example has been shown to dierentially aect gap junctional coupling be-
tween neonatal rat cardiomyocytes and A7r5 cells: low concentrations of oleic
acid (up to 1 mol/l) reduced dye coupling in A7r5 cells by 50%, but higher
92 7 Pharmacological Interventions at Gap Junctions
concentrations had no further eect. In contrast, neonatal rat cardiomyocytes
became uncoupled to zero levels in a linear fashion in concentrations ranging
from 1 to 25 mol/l [Hirschi et al., 1993].
Althoughoftenusedfor uncoupling, one shouldkeepinmindthat the eect
of these lipophilic agents is a physical eect on membrane structure. This means
that these drugs are valuable tools for investigation of single-channel behavior,
but theyare probablynot suitable for inducinguncouplinginorder toinvestigate
eects of putative coupling agents on the overall conductance g
j
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 K
d
of 4 mol/l and a Hill coecient of
0.75. The uncoupling was reversible g
j
returning back to 61% after 30 min
washout and recovery could be accelerated by addition of bovine serum albu-
min to the bath solution (g
j
reaching 86% of the initial value after 10 min of
washout). The eect was specic for arachidonic acid and could not be mim-
icked with analogues like arachidic acid (100 mol/l) or arachidonamide
(10 mol/l). The single-channel conductance
j
(mean
j
>33.5 pS) was not
aected by arachidonic acid at concentrations ranging from 1 to 100 mol/l,
so that the authors concluded that arachidonic acid might reduce the open
probability of the channel. In contrast, 100 mol/l arachidonic acid did not
aect nonjunctional membrane current in these experiments. 100 mol/l arach-
idonic acid induced uncoupling starting after 90 s, and after 2.5 min junctional
current was no longer detectable.
In addition to these ndings, Massey et al. [1992] reported that the uncou-
pling eect 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 mol/l U70344A, a 5-lipoxygenase inhibitor, whereas pretreatment
with the cyclooxygenase inhibitor indomethacin (100 mol/l) had no eect
on the arachidonic acid concentration-response curve. Complete uncoupling
occurred at membrane concentrations of 34 mol%. Incorporation of arachi-
donic acid into the lipid bilayer was not aected by the inhibitors. Complete
uncoupling was achieved with 20 mol/l arachidonic acid within about 3.5
min, with 5 mol/l within 4.5 min and with 2 mol/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 eect. However, lipoxygenase products like leuko-
trienes 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 eect.
93 7 Pharmacological Interventions at Gap Junctions
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 inter-
ventions, it was interesting that inhibtion of acylcarnitine transferase I by
10 mol/l POCAor by 100 mol/l oxfenicine completely prevented the accumu-
lation 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 inuence 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 ndings?
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 long-
chain acylcarnitines. According to the ndings 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 proba-
bility 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 nding that
arachidonic acid can reduce the conduction velocity of the action potential
[Bayer and Fo rster, 1979; Szekeres et al., 1976] may reect its uncoupling
action on g
j
. 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 arachi-
donic acid by inhibition of phospholipase A2 and the inhibition of acylcarni-
tine 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 eects 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 eectively uncouple gap junctions in craysh
septate axons lowering pH
i
to values around 6.2 [Peracchia, 1991a; Ramon et
al., 1991]. The uncoupling eect exhibits rapid onset and reversibilty. Similarly,
Nedergaard et al. [1995] reported on an uncoupling eect of 10 mmol/l lactic
acid adjusting the extracellular pH to values ranging between 6.48 and 7.30
in Hanks buered saline solutions. Lactic acid facilitates the intracellular
acidication under these conditions [Nedergaard et al., 1991]. Propionic
acid can also be used for uncoupling experiments as shown by Gottwald and
94 7 Pharmacological Interventions at Gap Junctions
Dhein [1997]. If considering the mechanisms of the induced uncoupling there
are two possibilities: rst, intracellular acidication may directly induce cellular
uncoupling via the pH
i
-uncoupling eect; 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 inuences 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 myo-
cytes. Using a silicon oil chamber Weingart [1977] demonstrated in cow hearts
that the exposure to 2 mol/l ouabain for 90 min increased longitudinal resis-
tance from 420 to 1,032 cm and concomitantly reduced the conduction
velocity from 50 to 29 cm/s. The increase in longitudinal resistance was associ-
ated with an increase in diastolic tension suggesting a rise in intracellular
calcium as the underlying mechanism. Similarly, De Mello [1976] observed
an uncoupling eect of 0.68 mol/l ouabain in Purkinje bers. He suggested
an increase in intracellular [Na
+
] and a secondary increase in intracellular
[Ca
2+
] 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 [Homan and Bigger,
1985] thereby decreasing the transmembrane sodium gradient which causes a
secondary rise in intracellular [Ca
2+
] via the impairment of the Na
+
/Ca
2+
-
exchange mechanism. Besides this an increase in the slow inward calcium
current I
si
has been described [Gilman et al., 1985]. Weingart and Maurer
[1987] studied the eects of exposing guinea-pig ventricular cell pairs to 2 and
20 mol/l strophanthidin. They found a dose- and time-dependent uncoupling
eect of strophanthidin on nexal resistance. 2 mol/l produced uncoupling
after 2025 min and 20 mol/l after 1015 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 mol/l strophanthidin from 19 to 295 M
in these experiments. Inhibiting the transmembrane calcium current I
si
antago-
nized the uncoupling eect of the cardiac glycoside indicating that extra Ca
2+
inux via I
si
contributes to the uncoupling action.
It can be imagined that these uncoupling eects 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
95 7 Pharmacological Interventions at Gap Junctions
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 accord-
ing to the ndings of De Mello [1976] who found a rapid uncoupling after
sodium injection.
Regarding manipulation of the intracellular calcium concentration,
caeine has been used in experiments on intercellular communication. If
cardiac cells are exposed to methylxanthines such as caeine a phasic release
of calcium from the sarcoplasmic reticulum can be observed [Chapman,
1979]. Maurer and Weingart [1987] investigated the eect of exposing adult
guinea-pig cardiac ventricular cell pairs to 510 mmol/l caeine. This inter-
vention did not change the gap junction conductance. However, if caeine
was applied after reduction of extracellular Na
+
(to 15 mmol/l, which reduced
g
j
by 20%), caeine induced a rapid (within 90 s) decrease in g
j
(74%). After
decoupling canine Purkinje cells by injection of calcium De Mello [1975]
found a slowed recovery in the presence of 6 mmol/l caeine (extracellular).
It is dicult at present to interpret these results from a pharmacological
point of view. Experiments have to be performed on the inuence of caeine
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 craysh septate axons that the uncoupling
eect of halothane could be enhanced by coadministration of caeine, 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 an-
ticipated that calmodulin antagonists should exhibit an inuence on gap junc-
tional coupling. Indeed, Peracchia et al. [1983] and Peracchia [1987] dem-
onstrated that calmodulin inhibitors were able to prevent cell uncoupling. In
craysh septate axon for example electrical uncoupling could be inhibited by
the calmodulin inhibitor W7 [Peracchia, 1987]. On this background systematic
studies on the inuence 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 eect of the calcium
channel antagonist, verapamil, on gap junctional conductivity. In the acinus
of the rat submandibular gland the uncoupling eect of the secretagogue
acetylcholine as assessed in dye-coupling studies could be inhibited in the
presence of 10 mol/l verapamil [Kanno et al., 1993]. This is probably due to
the antagonization of calcium inux. In control cells (without uncoupling by
acetylcholine) verapamil did not inuence cell coupling. Unfortunately, there
96 7 Pharmacological Interventions at Gap Junctions
are no data on the verapamil action on cardiac cells previously uncoupled by
any calcium agonistic process available.
Another possibility of inuencing gap junctional coupling is the modula-
tion 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 ki-
nases 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 fre-
quency distribution of unitary junctional conductance. Under the inuence
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 eect could be expected if phosphorylation of Cx43 would
be inhibited by an inhibitor of protein kinases. Such an inhibitor is staurospo-
rine which inhibits PKC and cyclic nucleotide-dependent protein kinases. Mo-
reno et al. [1994b] investigated the eect of 300 nmol/l staurosporine on single-
channel 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 modu-
lated pharmacologically. However, since the global gap junctional conductance
depends on single-channel conductance and open probability, the overall eect
on coupling cannot be concluded from these experiments. Because PKC has
been shown to increase g
j
[Kwak & Jongsma, 1996; Spray and Burt, 1990], it
can be anticipated that staurosporine, as an inhibitor of this enzyme, may
exhibit a decreasing eect on g
j
. On the contrary, the overall eect of okadaic
acid might consist of an increase in g
j
. However, more experiments will be
necessary for a nal 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 100160 nmol/l TPA, a stimulator of PKC, led to
a rapid decrease in the gap junction conductance g
j
. The onset of uncoupling
was observed 29 min after TPA application and maximum uncoupling within
24 min thereafter. They concluded that TPA may aect channel kinetics
rather than the single-channel conductance
j
. The TPA eect occurred only
acutely; 24-hour exposure of the cells to TPA did not result in changes in g
j
attributable to downregulation of PKC. The TPA-induced change in g
j
can
be prevented by pretreatment with the PKC inhibitor, staurosporine. In further
experiments Mu nster and Weingart [1993] showed that the TPA eect depends
97 7 Pharmacological Interventions at Gap Junctions
on the free intracellular calcium concentration: at low intracellular calcium
levels (18 nmol/l) the uncoupling TPAeect can be observed, whereas at higher
levels (100 nmol/l) the eect becomes mitigated or is completely inhibited (160
nmol/l). This nding is somewhat contradictory since it is well known that
PKC requires Ca
2+
for its action. However, the calcium sensitivity of the
various isoforms of PKC is dierent with PKC- exhibiting substantial activity
in the absence of calcium and an undened 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 eect 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 g
j
in response to TPA [Kwak
and Jongsma, 1996; Spray and Burt, 1990]. Kwak and Jongsma [1996] found
an increase by 162% in g
j
in neonatal rat cardiomyocytes after application
of 100 nmol/l TPA (intracellular calcium was buered with 10 mmol/l EGTA
in the pipette solution). TPA shifted the frequency distribution of unitary
conductances
j
to lower sizes. However, TPA decreased dye coupling in these
and other experiments [Kwak et al., 1995a].
The uncoupling TPAeects can also be mimicked with synthetic diacylgly-
cerol analogues such as 1-oleoyl-2-acetyl-glycerol [Mu nster and Weingart,
1993] in concentrations of 250 mol/l, which also activates PKC. Lower con-
centrations are ineective.
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. Takens-
Kwak and Jongsma [1992] investigated the inuence of 100 mol/l carbachol
on g
j
in cultured neonatal rat cardiomyocytes. In the whole cell technique
carbachol exposure decreased g
j
by 20%. The frequency distribution of unitary
currents was shifted by carbachol from 43 pS to a lower
j
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 aecting the other population of 20-pS channels. A similar decrease in
g
j
was obtained using 1.5 mmol/l 8-bromo-cGMP. The carbachol eect 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
observedreducedcouplinginresponse to100 mol/l carbachol inculturedadult
98 7 Pharmacological Interventions at Gap Junctions
rat and guinea-pig cardiomyocytes. However, uncoupling occurred only in the
presence of calciumwhereas in the absence of calciumcarbachol did not repress
dye coupling betweenthe cells. The uncoupling eect of carbachol has also been
establishedinother cells, for example pancreatic acinar cells [Somogyi andKolb,
1989] or rat submandibular gland [Kanno et al., 1993]. In the latter the eect 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 eects on
gap junctional coupling. Epinephrine has been shown to increase the spread
of electrotonic potentials during diastolic depolarization [De Mello, 1986b]
in canine Purkinje bers. This was interpreted as an eect of the rise in
intracellular cAMP resulting from-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 coup-
ling by the -adrenoceptor agonist isoproterenol in cardiac cell pairs. Thus,
stimulation of -adrenoceptors can be assumed to result in enhancement of
intercellular coupling, at least in some preparations. However, on the basis of
the ndings of Kwak and Jongsma [1996] on a lack of the eect 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 mol/l) has
been shown to decrease the percentage of dye-coupled cells [Kanno et al.,
1993], whereas isoproterenol was ineective, so that the authors concluded
that the mechanism was transmitted via action on the -adrenoceptors. This
was supported since the adrenaline eect could be suppressed by coadministra-
tion of 10 mol/l phenoxybenzamine.
Similar to a -adrenoceptor stimulation intracellular cAMP can be in-
creased 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], re-
sulting 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 eects as direct stimulation of PKC via phorbol esters. Indirect evidence
99 7 Pharmacological Interventions at Gap Junctions
for an improvement in cellular coupling by norepinephrine via
1
-adrenocep-
tors has been found in isolated rabbit hearts perfused according to the Langen-
dor technique, exposed to increasing concentrations of norepinephrine in the
absence and presence of the -blocker, propranolol, and the
1
-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 -adrenoceptors
by propranolol. The eect could be suppressed by prazosine. Since the enhance-
ment 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, angio-
tensin II was found to be eective in regulating cardiac gap junction conduct-
ance. In adult ventricular cell pairs De Mello [1992] observed a 55% reduction
in gap junction conductance g
j
within 20 s following the administration of
1 g/ml angiotensin II (approximately 0.9 mol/l) to the bath solution. The
angiotensin-II eect was reversible within 2.53 min. The concentration-re-
sponse curve started at 10 nmol/l resulting in a decrease of 18%. The uncoup-
ling angiotensin-II eect could be prevented by the PKC inhibitor
staurosporine and could be suppressed by DuP 753 (70 g/ml), an angiotensin
receptor-blocking agent, whereas DuP 753 alone did not alter g
j
. In additional
experiments De Mello [1992] investigated the inuence of the angiotensin-
converting enzyme inhibitor, enalapril (1 g/ml). Enalapril was added to the
bath solution and resulted in an increase of 106% within 45 min. The onset
of the eect took 1.5 min, probably the time needed in vitro for ester hydrolysis
of enalapril to MK-422, the active metabolite. The enalapril eect was dose-
dependent in the range from 0.25 to 1.25 g/ml, reaching maximum eect at
1.0 g/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 g
j
of 76% within 7 min. This could be completely inhibited by
intracellular dialysis of 1 nmol/l enalaprilat. Intracellular dialysis of angio-
tensin II led to a decrease in g
j
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 eect
could be prevented by intracellular administration of the receptor antagonist
DuP 753, De Mello [1994] concluded that there is an intracellular angiotensin-
II receptor involved in the regulation of g
j
.
Taken together these investigations point to a possible inuence 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 eects of
100 7 Pharmacological Interventions at Gap Junctions
angiotensin-converting enzyme inhibitors and protective eects 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 disper-
sion, this coupling eect of an angiotensin-converting enzyme inhibitor can
be seen as an antiarrhythmic action. In 11-month-old cardiomyopathic ham-
sters 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 immunouorescent staining [Dostal et al.,
1992] with the enzyme being located in the perinuclear region.
Another group of drugs aecting 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 g/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 Ca
2+
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 eective in vivo against CaCl
2
-induced and aconitine-induced arrhythmia
in mice [Kohama et al., 1987]. Moreover it prevented brillation 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 H
2
N-Gly-Pro-4Hyp-Gly-Ala-Gly-
COOH [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 CaCl
2
- 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 rst investigation directed toward the elucidation of the underlying mecha-
nism of action revealed that the antiarrhythmic peptide did not alter depolar-
101 7 Pharmacological Interventions at Gap Junctions
ization velocity, action potential amplitude, duration or shape and did not
exhibit any action on muscarinic receptors in canine Purkinje bers [Argentieri
et al., 1989], so that the authors concluded that the mechanism consisted of
eects 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 eects of the antiarrythmic peptide and
several synthetic derivatives, synthesized according to the Merrield technique
using the Fmoc strategy, in isolated rabbit hearts submitted to regional isch-
emia. 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 (H
2
N-Gly-
Ala-Gly-4Hyp-Pro-Tyr-CONH
2
), an amide, was found to be the most eective
with an onset of action at 0.1 nmol/l and maximum eect at 10 nmol/l. The
action consisted of a homogenization of the action potential duration so that
local dierences became smoothed without aecting the mean action potential
duration as shown in gure 22. The peptide did not exhibit any other inuence
on cardiac parameters (e.g. left ventricular pressure, coronary ow, QRS dura-
tion, PQtime). If hearts were submitted to regional ischemia by LADocclusion
for 30 min, pretreatment with 10 nmol/l AAP10 led to a signicant reduction
in the ischemia-induced alterations in the activation patterns and to a reduction
in the incidence of ventricular brillation, 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 eects of AAP10 on the transmembrane action potential in isolated papil-
lary muscles of guinea-pig heart. They found no eect on action potential
duration and morphology, on action potential amplitude, on maximum up-
stroke velocity or on resting membrane potential in concentrations up to
1 mol/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 propa-
gated action potential. The eect was reversible on wash out. Because due to
the lack of eect on the maximum upstroke velocity an eect on the sodium
current could be ruled out, the reduction in coupling time was a rst strong
hint of a possible action on the gap junctions. Thus, the authors decided to
investigate the eect 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 conguration).
In these experiments they found an improvement in g
j
by 10 nmol/l AAP10.
It took about 2 minutes until the onset of this eect. The eect could be
102 7 Pharmacological Interventions at Gap Junctions
Fig. 22. Reduction in dispersion of the ventricular action potential duration by the
synthetic antiarrhythmic peptide AAP10. The distribution of the action potential duration
(assessedas the epicardial activation-recoveryinterval, ARI) onthe surface of anisolatedrabbit
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].
103 7 Pharmacological Interventions at Gap Junctions
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 g
j
by 2.5 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 perfu-
sion, so that they uncoupled after 12 min. This could be prevented by pretreat-
ment with 10 nmol/l AAP10 [Dhein et al., 1997b; Mu ller et al., 1997b]. Since
in such low concentrations only a very slight eect 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
104 7 Pharmacological Interventions at Gap Junctions
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 inuenced by the intercellular axial resistivity in the
direction of propagation (R
i
) and is inversely dependent on the nonjunctional
sarcoplasmic membrane resistance (R
m
). As was described in the foregoing
chapters R
i
can be altered by a large number of factors and diseases. Changes
in cellular coupling can be expected to alter conduction velocity and synchro-
nization 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 eect and it is questionable whether enhancement of coupling
during manifest arrhythmia is more eective than the existing classic antiar-
rhythmics. In situations with uncoupling being due to structural changes such
as brosis, drugs which can improve gap junctional coupling are probably
ineective 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 eective in the prophylaxis of
ischemia-associated ventricular brillation (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 antiar-
rhythmic peptides are probably not the nal 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 rst step in the direction of a newclass of drugs inuencing gap junctional
coupling.
The various pharmacological approaches to the modulation of gap junc-
tional coupling are summarized in gure 23.
105 7 Pharmacological Interventions at Gap Junctions
8
...........................
Methods for Investigation of
Gap Junctions
In this chapter more detailed information on the double-cell voltage-
clamp 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 dierent 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 authors laboratory) will be discussed below.
8.1 Culture of Neonatal Rat Cardiomyocytes
Prepare the following solutions:
Coating solution
M199 (with Earls salts)
100 g/ml penicillin
100 g/ml streptomycin
10% fetal calf serum (FCS)
PBS/glucose solution
NaCl 137 mmol/l
KCl 2.7 mmol/l
Na
2
HPO
4
8.3 mmol/l
KH
2
PO
4
1.5 mmol/l
Glucose 20 mmol/l
pH to be adjusted to 7.4
Desaggregation solution
Phosphate-buered saline (PBS) 50 ml
Glucose 200 mg
Bovine serum albumin (BSA) 500 mg
Collagenase type II (Gibco) 50 mg (204 U/mg)
106
Medium for resuspension of supernatants
M199 (with Hanks salts and HEPES)
100 g/ml penicillin
100 g/ml streptomycin
10% FCS
25 mmol/l HEPES
2 mmol/l L-glutamine
Culture medium for the 1st day
M199 (with Earls salts and HEPES)
2 mmol/l L-glutamine
5% FCS
100 g/ml penicillin
100 g/ml streptomycin
Culture medium after the 1st day
M199 (with Earls salts and HEPES)
2 mmol/l L-glutamine
1% FCS
100 g/ml penicillin
100 g/ml streptomycin
Dulbeccos wash solution for the 2nd day
NaCl 137 mmol/l
KCl 2.68 mmol/l
Na
2
HPO
4
2H
2
O 6.48 mmol/l
KH
2
PO
4
1.47 mmol/l
MgCl
2
6H
2
O 0.49 mmol/l
MgSO
4
7H
2
O 0.81 mmol/l
CaCl
2
0.9 mmol/l
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 (12 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 C. 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 89 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-cm
2
plastic asks and incubate at 37 C/5% CO
2
.
After 2 h of preplating (this time is required for nonmuscular cells to attach) the supernatant
of this askis lteredthrougha nylonmesh(pore width100 m) andthenseededinPetri dishes
at a density of about 10,000 to 100,000 cells/cm
2
in culture medium for the 1st day. After 24 h
the Petri dishes are rinsed o with Dulbeccos wash solution and culture medium for the 1st
107 8 Methods for Investigation of Gap Junctions
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 broblast 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 authors laboratory. Besides this, other protocols
may also suit.
Prepare the following solutions:
Glutamine solution
Glutamine 200 mmol/l
Hanks buered saline solution (HBSS)
NaCl 8,000 mg/l
KCl 400 mg/l
KH
2
PO
4
60 mg/l
Na
2
HPO
4
47.5 mg/l
NaHCO
3
350 mg/l
Glucose 1,000 mg/l
pH 7.4
Trypsin solution (0.25%)
Trypsin 2.5% in HBSS (+phenol red) 3 ml
HBSS 27 ml
Culture medium
FCS 4%
Horse serum 2%
Glutamine solution 3.4 ml/l
Penicillin 100 IU/ml
Streptomycin 100 IU/ml
M199 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 (57 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 eggs pole under sterile
conditions in laminar ow. Decapitate the embryo, isolate the heart from the embryo and
remove the atria. Bath the ventricles in 510 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 C for 7 min. Thereafter mix the solution well
and lter through gauze with a mesh size of 100 m in a Falcon tube containing 10 ml HBSS
(4 C) and 4% FCS. Centrifuge for 5 min at 1,500 rpm and 4 C. Resuspend the pellet with
HBSS, centrifuge again and repeat the procedure once again. Dissolve the pellet in about 10 ml
108 8 Methods for Investigation of Gap Junctions
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 asks 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 desir-
able to yield a high amount of cell pairs. Therefore, protocols using proteases such as trypsin
shouldnot beused, becauseproteases mayenhanceseparationof thecells anddisrupt 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
authors laboratory a collagenase protocol is commonlyusedtoisolate adult guinea-pigcardio-
myocyte 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 21 ml
3 mol/l KCl 783 l
0.1 mol/l KH
2
PO
4
6 ml
0.1 mol/l MgSO
4
12.5 ml
0.4 mol/l NaHCO
3
31.1 ml
0.5 mol/l HEPES/Na 5 ml
Glucose 0.54 g
H
2
O add 500 ml
Solution B
Pyrovate 44 mg
BSA 200 mg
Solution A add 200 ml
Equilibrate with 95% O
2
and 5% CO
2
, adjust pH to 7.4
Solution C
1 vial collagenase Worthington type 100 U/ml
12.5 l 0.1 mol/l CaCl
2
Solution B add 10 ml
Solution D
Solution B 20 ml
0.1 mol/l CaCl
2
10 ml
Equilibrate with 95% O
2
and 5% CO
2
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 l
M199 add 20 ml
109 8 Methods for Investigation of Gap Junctions
After the guinea pig (250350 g) has been killed, the heart is prepared according to
the Langendor technique and transferred to a Langendor apparatus and perfused at 37 C
for 10 min with solution B (i.e. Ca
2+
-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% O
2
and 5% CO
2
at 37 C.
Next, the solution containing the tissue is ltered through gauze with a mesh size of
200 m and centrifuged for 1 min at about 600 rpm. After removing the supernatant, the
pellet is resuspended in 10 ml solution D (37 C; 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 l
0.1 mol/l CaCl
2
, 7.5 l after another minute, 7.5 l after the next minute and 10 and 15 l
in the following 2 min, thereby adjusting the calcium concentration nally 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 Ca
2+
) 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, immunohisto-
chemical methods are commonly used. The best results are obtained with frozen sections,
since other methods such as paran embedding or acrylate embedding may aect the
antigeneity of the proteins to be detected (this is because tissue xation, for example, glutaral-
dehyde 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 C) 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-buered solution (e.g. Tyrodes solution) and
glycerine (1:1)
Let the uid drain
Transfer the heart to a methanol bath (pre-cooled to 70 C)
Let the methanol drain
Transfer the tissue to uid N
2
110 8 Methods for Investigation of Gap Junctions
In the next step sections have to be prepared fromthe tissue block. Frozensections should
be cut at 5-m thickness and be picked on aminopropyltriethoxysilane (APES)-coated slides
(alternative: Superfrost slides) using a cryostat at 25 C. 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 xed in acetone for 20 min (alternatively in methanol,
30 min). Thereafter, sections are incubatedfor 30 mininTritonX100(0.1%; this stepis option-
ally, but canbeusedtopermeabilizethetissueor culturedcells if theantibodybinds tointracellu-
lar-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 unspecic
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 252270; 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 specic 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 FITC-
coupled 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 FITC-
labeled goat anti-mouse IgGantibody, Sigma was used in the authors laboratory. Using uor-
escence microscopy is a very elegant procedure which should be preferred if possible. However,
in some cases problems may arise from unspecic uorescence or, for certain investigations,
fromthe 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 unspecic 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 C (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 >470 nm; excitation lters: 2SP490+2 mmLP455) and emit green uor-
escent light (at >540 nm; emission lter; barrier lter; LP 515).
Protocol for immunostaining (as used in the authors 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 C and seal with acryl varnish
PBS: 8 g NaCl, 0.2 g KCl, 1.15 g Na
2
HPO
4
, 0.2 g KH
2
PO
4
, add H
2
O to a nal 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 anti-
bodies and for the streptavidin-biotin system are given [Jackson and Blythe, 1993; Ormerod
and Imrie, 1989].
111 8 Methods for Investigation of Gap Junctions
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-buered saline (TBS)
Let the uid drain
Incubate with primary antibody in TBS for 1 h
Wash with TBS (20 min, 3 times)
Let the uid 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 l streptavidin, 20 l bio-
tinylated horseradish peroxidase, 1 ml TBS (shake well and wait 30 min)
Let the uid drain from the sections and incubate with the streptavidin-biotin/horseradish
peroxidase complex for 3060 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 H
2
O, pH
is adjusted to 7.6 with HCl, 85 g NaCl is added with H
2
O to a nal volume of 10 liters
DAB stock solution: 7.5 g DAB in 300 ml Tris buer at pH 7.6 (caution, DAB is carcino-
genous)
Copper sulfate solution: 4 g CuSO
4
, 7.2 g NaCl, add to 1 liter H
2
O
For peroxidase-coupled secondary antibody the following protocol may be used:
Air-dryed frozen sections have to be rehydrated in TBS
Let the uid drain
Incubate with primary antibody in TBS for 1 h
Wash with TBS (20 min, 3 times)
Let the uid drain
Block the endogenous enzyme with 0.3% H
2
O
2
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
112 8 Methods for Investigation of Gap Junctions
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 anti-
alkaline 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 uid drain
Incubate with primary antibody in TBS for 1 h
Wash with TBS (20 min, 3 times)
Let the uid 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 buer (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 nd the most suitable for his purposes by varying the
several steps. However, some problems often encountered with immunohistology will be
discussed briey. The antibodies may bind to unspecic 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 12% 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 3,0006,000) to the
diluted antibody. Disturbing autouorescence may be reduced by staining the tissue with
pontamine sky blue (0.05% in PBS with 1% dimethylsulfoxide) for 30 min before applying
the rst 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 nonspecic reactions
of the second antibody. In addition, if immunouorescence is used, unstained slides have to
be investigated also as controls in order to detect nonspecic background uorescence (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 specic connexin
within the tissue or within cells. It can also be used for semiquantitative evaluations by
113 8 Methods for Investigation of Gap Junctions
counting, for example, the percentage of positively stained cells among all cells in a view
eld, 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 elds, and
will then give reliable results. A quantication of the protein content by measuring the
intensity of staining (e.g. of immunoourescence) 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 immunouorescence is extremely error
prone and dicult, and great caution has to be taken in order to garantee that there is no
dierence in bleeching between the samples, the optical system is unchanged, the background
is corrected exactly in the same way and so on. Thus, if quantication 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 authors 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 04 C if not stated otherwise.
1 Transfer tissue (heart) to cold 1 mmol/l NaHCO
3
(pH 8.2)
2 Remove supercial fat and vessels
3 Cut the tissue into small pieces and add these to 50 ml 1 mmol/l NaHCO
3
(pH 8.2)
4 Add phenylmethylsulfonyl uoride (PMSF) to a nal PMSF concentration of 1 mmol/l
5 Stir 15 min at 4 C
6 Homogenize tissue: 60 s, V
max
(Virtis homogenizer)
7 Further homogenization using tissue mizer, SDT 100 EN
8 Dilute the homogenate to 300 ml with 1 mmol/l NaHCO
3
(pH 8.2), and lter through
68 sheets of medical gauze
9 Centrifuge the ltrate: 15 min, 33,000 g
10 Remove the supernatant and disolve the resulting pellet in 300 ml 1 mmol/l NaHCO
3
(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 Na
2
S
2
O
3
in
1 mmol/l NaHCO
3
(pH 8.2)
13 Add PMSF (as step 4)
14 Stir overnight at 4 C
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 Na
2
S
2
O
3
in
1 mmol/l NaHCO
3
(pH 8.2), homogenize in homogenizer (3 strokes only)
114 8 Methods for Investigation of Gap Junctions
18 Centrifuge: 30 min, 27,000 g
19 Remove supernatant, wash pellet in 70 ml (nal volume) 0.6 mol/l KI, 6 mmol/l Na
2
S
2
O
3
in 1 mmol/l NaHCO
3
(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 C
28 Take the band at the 3544.5% 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 C
31 Take the band at the 3544.5% 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 3544.5% interface and dissolve 1:1 in 0.3% deoxycholate in
5 mmol/l Tris (pH 10)
34 Centrifuge: 30 min, 106,000 g, 15 C
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 C
39 Take the material at the 31.535% interface
40 Centrifuge: 30 min, 106,000 g, 15 C
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 authors 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
H
2
O: 2.6 ml
115 8 Methods for Investigation of Gap Junctions
Ammonium persulfate: 10% (w/v) in water: 30 l
N,N,N,N-tetramethylethylenediamine (TEMED): 6 l
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
H
2
O: 5.5 ml
Ammonium persulfate: 10% (w/v) in water: 150 l
TEMED: 30 l
Sample buer
0.5 mol/l Tris (pH 6.8) 1.2 ml
20% SDS in H
2
O 1 ml
Glycerin 1 ml
5% -Mercaptoethanol
0.1% (v/v) bromphenol blue in ethanol 0.5 ml
The sample buer is distributed in Eppendorf tubes in amounts of 500 l. Finally, 25 l -
mercaptoethanol is added prior to use.
Running buer
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 H
2
O
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-l samples.
Fill the indentations with running buer and add 40 l of sample (the sample is dissolved
1:1 in sample buer). 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 C 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
116 8 Methods for Investigation of Gap Junctions
up the experiment, because for these two kinds of investigation, dierent ampliers 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 dicult or
even impossible to accurately measure the voltage dependence of gap junctions.
This series resistance problem can be avoided by the use of discontinuous single-
electrode voltage-clamp (dSEVC) ampliers or switch-clamp ampliers. These ampliers
switch between voltage measurement and current injection thereby avoiding artefacts by the
resistance of the pipettes. The use of switch-clamp ampliers 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 ampliers is a bit more complicated than that of
normal patch-clamp or voltage-clamp ampliers, 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 ampliers 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 ampliers to measure single-channel events.
In the authors laboratory gap junction conductance between two cells is measured
using the double-cell voltage-clamp method with two switch-clamp ampliers (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 authors laboratory) consists of the
following components:
2 SEVC ampliers
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 ampliers to the computer)
1 PC system equiped with an A/D converter
Software for controlling the SEVC ampliers (make sure that your software can control two
ampliers; 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
ampliers, 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
117 8 Methods for Investigation of Gap Junctions
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-lled 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 23 M
resistance, re-polish in a microforge or treat with sylgard. Prepare the following solutions.
Extracellular solution
NaCl 135 mmol/l
KCl 4 mmol/l
CaCl
2
2 mmol/l
MgCl
2
1 mmol/l
NaH
2
PO
4
0.33 mmol/l
HEPES 10 mmol/l
Glucose 10 mmol/l
pH 7.4
Pipette solution (intracellular solution)
CsCl 125 mmol/l
NaCl 8 mmol/l
CaCl
2
1 mmol/l
EGTA 10 mmol/l
Na
2
ATP 2 mmol/l
MgATP 3 mmol/l
Na
2
GTP 0.1 mmol/l
HEPES 10 mmol/l
pH 7.2 (with CsOH)
This solution should be ltered through a sterile lter 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 (2124 C) or at
37 C as desired. Adjust the ampliers and compensate for series resistance or capacity. If
SEVC ampliers are used adjust the switching frequency to values of about 2530 kHz.
Make sure that the command potential is reached within 35 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 G) for one cell, perform the breakin
and achieve the whole cell conguration by applying a sharp suction pulse. Thereafter try
to establish a gigaseal and afterwards a whole-cell conguration in the other cell. 35 min
after establishing the whole-cell conguration the experiment is started. Sampling frequency
is adjusted to 10 kHz and the obtained signal is low-pass ltered at 1 kHz. During the
recordings 1 mmol/l BaCl
2
is added to the external solution.
One can use either a symmetrical or an asymmetrical protocol. First both cells are
clamped to 40 mV holding potential in order to inactivate the sodium current. Thereafter,
one cell is clamped to potential ranging from 90 to +10 mV for 200 ms (pulse duration;
asymmetrical protocol). Thereby, a transcellular voltage dierence of 50 mV can be applied
118 8 Methods for Investigation of Gap Junctions
Table 4. Dyes used for investigation of dye coupling
Dye Excitation Emission Molecular
wavelength weight
(nm)
Lucifer yellow 430 535 457
Procion yellow 488 530 697
2,7-Dichlorouorescein 513 532 691
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 owing 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 80 and 40 mV.
Measurements should be done in both cells alternatively.
For a symmetrical protocol both cells are clamped to the common holding potential
of 40 mV. Thereafter, both cells are pulsed equally but with opposite polarity. Thus, cell
1 is clamped to 50 mV and cell 2 to 30 mV, then to 60 and 20 mV, respectively,
and so forth.
If voltage dependence is to be investigated the pulse duration must be enhanced to
values of 12 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 g
j
in these
preparations, for example in neonatal rat heart cells Schmilinsky-Fluri et al. [1990] found a
decrease in g
j
of 16.4% in 6 min which could be antagonized by addition of a phospholipase
inhibitor, 20 mol/l bromophenacyl bromide, to 1.8% within 6 min. They suggested that
endogenous arachidonic acid is involved in spontaneous uncoupling. Others favored a wash-
out 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 dier
from each other in certain situations. For dye-coupling studies, LY is dissolved in water at
concentrations of 35%. The cell is penetrated with a LY-lled pipette and stained by
application of constant hyperpolarizing current pulses of 410 nA (1 s pulse duration, 0.5
Hz frequency) for 12 min. LY-lled 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 0.10.5%. After establishing the whole cell conguration, the
119 8 Methods for Investigation of Gap Junctions
cell is clamped to negative potential for 15 min in order to increase the intracellular dye
concentration.
Staining of the injected cell and the adjacent cells can be observed directly using an
epiuorescence microscope (table 4). The excitation light is emitted from a mercury high-
pressure lamp and ltered by a band-pass excitation lter (BP 450490). The light then
passes a dichroic mirror (FT 510) which serves as a beam splitter. Light with wavelengths
of =510 nm is selectively reected by the mirror, whereas longer wavelengths pass it. Thereby
excitation light of 450510 nm reaches the specimen and excites the dye. The light emitted
by the dye passes the beam splitter and is ltered by a long-pass emission lter (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. Neouar, Zeiss).
Acknowledgements
I greatly thank Dr. Aida Salameh for the superior help with the gures, Mrs. Michaela
Gottwald for intensive corrections and help with the search for literature, Mrs. Kathi Kru se-
mann for taking the histological photographs, and Mr. Rajiv Grover for language revision.
120 8 Methods for Investigation of Gap Junctions
...........................
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...........................
Appendix
One-letter code of amino acids, three-letter code and codons (53) coding for the
amino acid
Amino acids with apolar side chains
A Ala Alanine GCU, GCC, GCA, GCG
V Val Valine GUU, GUC, GUA, GUG
L Leu Leucine CUU, CUC, CUA, CUG, UUA, UUG
I Ile Isoleucine AUU, AUC, AUA
P Pro Proline CCU, CCC, CCA, CCG
F Phe Phenylalanine UUU, UUC
W Trp Tryptophane UGG
M Met Methionine AUG
Amino acids with uncharged polar side chains
G Gly Glycine GGU, GGC, GGA, GGG
S Ser Serine AGU, AGC, UCU, UCC, UCA, UCG
T Thr Threonine ACU, ACC, ACA, ACG
C Cys Cysteine UGU, UGC
Y Tyr Tyrosine UAU, UAC
N Asn Asparagine AAU, AAC
Q Gln Glutamine 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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1125 Dixwell Avenue
Hamden, CT 06514 (USA)
Tel. +1 203 776 0664
Fax +1 203 776 1278
(glass, AD/DA converter, ampliers and
equipment)
WPI World Precision Instruments
Harry Fein
Liegnitzer Strasse 15 D10999 Berlin
(Germany)
Tel. +49 30 618 8845
Fax +49 30 618 8670
(microscopes, oscilloscopes and
electrophysiological equipment)
Carl Zeiss Jena
Tatzendpromenade 1a
D07740 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 elds 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 List of Suppliers of Specialized Items
..............................
Subject Index
AAP10, gap junction uncoupling 102, ATP, gap junction channel regulation
44, 45 104, 105
Acetic acid, gap junction uncoupling 94 Atrioventricular bundle, gap junction
distribution 28, 30 Acetylcholine, gap junction channel
regulation 46 Atrioventricular node, gap junction
distribution 27, 29, 30 Aconitine, gap junction uncoupling
95, 96 Atrioventricular reentry 9
Atrium, gap junction distribution 29 Action potential propagation
changes in arrhythmia 12
coupling eects on transverse and Bundle branches, gap junction
distribution 30 longitudinal propagation 5, 6
eects of nonuniformity 4, 5
Acute cardiac disease, eects on Cable properties, muscle bers 3, 4
Caeine, eect on uncoupled gap gap junctions 7378
Acylcarnitines, gap junction uncoupling junctions 96
Calcium, gap junction channel regulation 7476, 94
Adrenaline, gap junction channel 37, 38, 4043
cAMP, see Cyclic AMP regulation 47, 99
Aging, eects on gap junctions 88 Capacitive coupling 2, 3
Carbachol, gap junction uncoupling 98, 99 Angiotensin II, gap junction uncoupling
100, 101 Carbon dioxide, see pCO
2
Cardiac arrhythmia suppression trial Anisotropic ratio, regional variations 6
Anisotropic reentry 9, 11 (CAST), proarrhythmic risk of
antiarrhythmic drugs 1 Antiarrhythmic peptides 101, 102,
104, 105 Cardiac myocyte
culture Arachidonic acid, gap junction channel
regulation 45, 46, 93, 94 embryonic chick cardiomyocytes
108, 109 Arrhythmia
eects on gap junctions 83, 84 neonatal rat cardiomyocytes 106108
broblast-myocyte gap junctions 33 types in ischemia 10, 11, 74
Arrhythmogenic substrate, denition 1, 2 isolation from guinea pig 109, 110
shape eects on propagation 7, 8 Assembly, gap junction channel 6469
145
CAST, see Cardiac arrhythmia equations 51, 52
voltage relationships in gap suppression trial
Cell culture junction channels 5254
Cyclic AMP (cAMP), gap junction channel electrophysiology, systems for study 50
embryonic chick cardiomyocytes regulation 3538, 69, 70
108, 109
neonatal rat cardiomyocytes 106108 Decanoic acid, gap junction uncoupling 91
Defective heart development, eects Chagas disease, eect on gap junctions
8486 on gap junctions 86, 87
Degradation, gap junction channel 68, 69 Chronic ischemic heart disease, eects
on gap junctions 11, 7981 Distribution, see Gap junction distribution
Double-cell voltage-clamp technique Connexins
assembly 6669 50, 51, 116119
Dye-coupling studies 119, 120 assembly of gap junctions 6466, 68
Cx40 gene mutations 87
Cx43 Electric eld coupling 3
Electron microscopy, gap junction gene
mutation in defective heart distribution 25, 26, 34
Electrophysiology, gap junction channels development 86, 87
regulation 71, 72 cell systems for study 50
connexins isolation 114116
physical properties 14, 15 Cx26 single-channel conductance 61
Cx37 single-channel conductance isoform expression in gap junctions
6, 13 60, 61
Cx40 single-channel conductance multiple protein channels 34
posttranslational modications 59, 60
Cx43 single-channel conductance 59 1416, 40, 66, 69
sequences 20, 21 Cx45 single-channel conductance 60
transjunctional voltage sensitivity single-channel conductance
Cx26 channels 61 5557
current Cx37 channels 60, 61
Cx40 channels 59, 60 components 52
equations 51, 52 Cx43 channels 59
Cx45 channels 60 voltage relationships 5254
double-cell voltage-clamp technique species variability 23, 24
staining and distribution 2731, 110114 50, 51, 116119
intercellular resistance 52, 54, 58, 59 structure 13, 14
synthesis 6466, 68, 69, 71, 72 ion permeability 61, 62
open probability of a single channel 57 transjunctional voltage sensitivity 5557
turnover 68, 69 states of single-channel conductance 50
types of channels 50 types 13, 1923
Coronary vasculature voltage sensitivity of channels 56, 57
Enalapril, eect on uncoupled gap chronic ischemic heart disease,
eect on gap junctions 81 junctions 100, 101
Endothelial cells, gap junction gap junction distribution 31
Current distribution 31
Ethrane, gap junction uncoupling 90 components 52
146 Subject Index
Excitation, transfer between cardiac laser scanning confocal microscopy
27, 34 cells 2, 3
multiple connexin channels 34
myocyte-broblast junctions 33 FGF-2, see Fibroblast growth factor-2
Fibroblast growth factor-2 (FGF-2) Purkinje bers 30
sinoatrial node 29 gap junction channel regulation 48, 71
release in chronic ischemic heart topology 26, 27
ventricular myocardium 30, 31, 34 disease 79
Gap junction channel Halothane, gap junction uncoupling 91
Heart failure, eects on gap junctions assembly 6469
cardiac disease-induced changes 82, 83
Heptanol, gap junction uncoupling 90 acute cardiac disease 7378
aging 88 Hypertension, eect on gap junctions 83
arrhythmia 83, 84
chronic ischemic heart disease Immunostaining
connexins 2731 7981
defective heart development 86, 87 protocol 110114
Infective heart disease, eects on heart failure 82, 83
infective heart disease 8486 gap junctions 8486
Ion permeability, gap junction closure mechanism 18
clustering 17 channels 61, 62
Ischemia degradation 68, 69
developmental expression 63, 66 associated arrhythmias 10, 11
gap junction changes electrophysiology, see Electro-
physiology, gap junction channels acute cardiac disease 7378
chronic ischemic heart disease functions, overview 13, 25, 4850
new channel formation, kinetics 63, 64 11, 7981
tissue properties in healing phase 11 regulation, see specic regulators
structure 13, 16, 17
synthesis 6472 Laser scanning confocal microscopy,
gap junction distribution 27, 34 uncoupling
drugs, see specic drugs Leading circle concept 8
eects in heart disease 77, 78, 89, 90
Gap junction distribution Magnesium, gap junction channel
regulation 44 atrioventricular bundle 28, 30
atrioventricular node 27, 29, 30 Myocyte, see Cardiac myocyte
atrium 29
bundle branches 30 Noradrenaline, gap junction channel
regulation 47, 99 chronic ischemic heart disease eects
89, 81
connexin staining 2731, 110114 Octanol, gap junction uncoupling 90
Oleic acid, gap junction uncoupling 92, 93 coronary vasculature 31
eects on conduction 9, 10, 25 Ouabain, gap junction uncoupling 95
electron microscopy 25, 26, 34
endothelial cells 31 Palmitoleic acid, gap junction
uncoupling 91 heart failure eects 82
147 Subject Index
pCO
2
, gap junction channel regulation Protein kinase G (PKG), gap junction
channel regulation 39, 40 43
Permeability, gap junction channels to Purkinje bers, gap junction
distribution 30 ions 61, 62
pH, gap junction channel regulation
42, 43 Resistance, gap junction channels 52,
54, 58, 59 Phospholipase C (PLC), gap junction
channel regulation 38
Phosphorylation, gap junction channel Sinoatrial node, gap junction
distribution 29 regulation 40, 97
PKA, see Protein kinase A Sodium, gap junction channel
regulation 44 PKC, see Protein kinase C
PKG, see Protein kinase G Strophanthidin, gap junction
uncoupling 95 PLC, see Phospholipase C
Potassium eux, acute cardiac disease Synthesis, gap junction channel 6472
73, 74
Propagation velocity, ischemia eects Temperature, gap junction channel
regulation 35 75, 76, 90
Prophylaxis, proarrhythmic risk of Tyrosine kinase, gap junction channel
regulation 40, 70, 71 antiarrhythmic drugs 1, 2
Propionic acid, gap junction uncoupling
94, 95 Uncoupling, gap junction channel
drug induction 90105 Protein kinase A (PKA), gap junction
channel regulation 3539 eects in heart disese 77, 78, 89, 90
Protein kinase C (PKC), gap
junction channel regulation 38, 39, 46, Ventricular myocardium, gap junction
distribution 30, 31, 34 70, 97100
148 Subject Index