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864
Reduced Content of Connexin43 Gap Junctions
in Ventricular Myocardium From Hypertrophied
and Ischemic Human Hearts
N.S. Peters, MRCP; C.R. Green, PhD; P.A. Poole-Wilson, MD, FRCP; N.J. Severs, PhD
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Background.Gap junctions are a determinant of myocardial conduction. Disturbances of gap-junctional
content may account for abnormalities of impulse propagation, contributing to the arrhythmic tendency
and mechanical inefficiency of ischemic and hypertrophied myocardium. The aim of this study was to
characterize gap junction organization in normal human ventricular myocardium and to establish
whether abnormalities exist in myocardium of chronically ischemic and hypertrophied hearts.
Methods and Results. Cardiac gap-junctional connexin43 antibodies and confocal microscopy were used
in a quantitative immunohistochemical study of surgical myocardial samples to explore the structural
basis of electromechanical ventricular dysfunction in chronic ischemic and hypertrophic heart diseases.
Normal adult human left ventricular myocardium had a gap-junctional surface area of 0.0051 pm2/pm3
myocyte volume; gap junctions were confined to intercalated disks, of which there was a mean of 11.6 per
cell. The right ventricle showed similar gap junction surface area. Left ventricular myocardium from
ischemic hearts (distant from any fibrotic scarring), despite normal numbers of intercalated disks per
cell, had a reduced gap junction surface area (0.0027 Ftm2/'lm3; P=.02), as did hypertrophied
myocardium (0.0031 tm2/pm3; P=.05). The cardiac myocytes in the pathological tissues were larger than
normal, and estimated gap-junctional content per cell was reduced in ischemic ventricle (P=.02)
compared with normal.
Conclusions. Gap junctions in normal adult human working ventricular myocardium occupy an area of
0.0051 am2/,'lm3 myocyte volume. This surface area is reduced in ventricular myocardium from hearts
subject to chronic hypertrophy and ischemia, despite a normal number of intercellular abutments, and
this alteration may contribute to abnormal impulse propagation in these hearts. (Circulation.
1993;88:864-875.)
KEY WORDS *
gap junctions
*
structure
*
G ap-junctional organization is an important determinant of intercellular conductance and
the conduction properties of myocardium.1-4
The normal pattern of anisotropic conduction in ventricular myocardium, by which conduction parallel to
the myocyte long axis is up to four times more rapid
than that transverse to it,5,6 is dependent in part on the
low resistivity of the gap-junctional membranes, their
distribution, and their abundance.
The underlying mechanism of many clinical cardiac
arrhythmias is a myocardial reentrant electrical circuit,7
perpetuation of which requires areas of slowed conduction within the circuit, so that the advancing wave front
encounters excitable tissue78 and initiation of which is
enhanced by a local dispersion of recovery of excitability of the constituent myocytes.9-11 These factors are
thought to provide the substrate for clinical reentrant
arrhythmias.12 Efforts in basic cardiac electrophysiology
have focused on how alterations of active membrane
ionic properties can lead to slowing of electrical propReceived April 23, 1992; revision accepted May 12, 1993.
From the National Heart and Lung Institute (N.S.P., P.A.P.-W.,
N.J.S.) and University College (C.R.G.), London, England.
Correspondence to Dr N.S. Peters, Department of Cardiac
Medicine, National Heart and Lung Institute, Dovehouse Street,
London SW3 6LY, England.
hypertrophy
*
ischemia
*
microscopy
agation and affect recovery of excitability, but this
approach has not provided a full explanation of arrhythmogenesis. Indeed, there are reports of normal membrane potential characteristics in myocytes from myocardium with markedly abnormal electrophysiological
properties and manifest arrhythmias.13-15 There has
been increasing recognition that changes in the passive
resistivity encountered by a propagating impulse can
reduce conduction velocity and increase heterogeneity
of conduction, irrespective of changes in active membrane properties.16-19 Despite the recognition of this key
influence on the behavior of the propagating impulse,
little information is available on the quantity and distribution or the function of gap junctions in human
myocardium in health or disease.
In this study, immunohistochemical detection of the
principal cardiac gap-junctional protein, connexin43, by
confocal laser scanning microscopy and electron microscopic techniques were used to examine the structural
organization of gap junctions in normal human ventricular myocardium and to establish whether changes in
gap junction content and organization occur in diseased
ventricular tissue in which there are recognized alterations of myocyte form and electromechanical interaction. The results provide new insights into a possible
Peters et al Gap Junction Quantification in Human Myocardium
anatomic factor in the pathogenesis of electromechanical dysfunction and arrhythmias.
Methods
Myocardial Samples
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Ventricular specimens were obtained from patients
fulfilling the study criteria (defined below), selected at
random from those undergoing cardiac surgery having
given prior written consent, or from transplant donor
hearts. The biopsies were taken from either the left
ventricular apex or the mid right ventricular free wall as
appropriate in each case, either by excision of a 3-mm
ellipse of epimyocardium or by use of a transmural
needle bioptome (diameter, 1.5 mm) from which the
epimyocardial end was used for study. All biopsies
obtained at routine surgery were taken immediately
after the heart was arrested on cardiopulmonary bypass.
Normal ventricular myocardium. Completely normal
human myocardium for immediate fixation is not available for experimentation. For the purposes of this study,
"normal" myocardium was obtained from the following
sources:
1. Cardiac transplant donor hearts from which a
single right ventricular biopsy was obtained on arrival at
the implanting hospital after the heart was transported
in cardioplegic solution on ice (for up to 4 hours). Light
and electron microscopy have established that no detectable disruption of the intercalated disk structure
occurs under these conditions of cardioplegia, a finding
consistent with separate studies on myocardial preservation and cardioplegia.20 Samples of both ventricles of
normal donor hearts that for technical reasons were not
used for transplantation as intended were also studied.
2. The left ventricular apical region from patients
with the Wolff-Parkinson-White syndrome (WPW).
This group of patients experienced occasional but symptomatic tachyarrhythmic episodes but had no other
detected cardiac dysfunction on routine preoperative
assessment and were undergoing surgical division of the
accessory pathway. Ventricular myocardium from
hearts such as these, with infrequent arrhythmic episodes, has not been shown to have any structural
abnormality and is generally considered normal.21
By these criteria, five "normal" left ventricular samples (two transplant donor hearts and three WPW
hearts) and five right ventricular samples (transplant
donors) were studied.
Myocardium from ischemic ventricle. Left ventricular
myocardium was obtained from five patients with threevessel coronary artery disease undergoing coronary
artery bypass graft surgery for symptomatic relief from
angina. All patients had had documented anterior myocardial infarction (with or without Q waves on ECG) at
least 3 months previously and were normotensive. Preoperative `1Tl perfusion scintigraphy demonstrated
symptomatic exercise-induced reversible ischemia in the
remainder of the anteroapical territory, from which the
biopsies of viable epimyocardium were obtained in each
case. Myocardium immediately abutting scar from past
infarction, shown in an earlier study22 to have a highly
disrupted gap junction distribution (to a mean distance
of 123 1um from the scar tissue border), was not included
for analysis in the present study. For the same reason,
subendocardial myocardium, which may contain wide-
865
spread small scars in patients with chronic coronary
disease,23 both disrupting gap-junctional distribution
and distorting tissue architecture, was also avoided. This
strategy allowed sampling of viable myocardium associated with a region of recurrent reversible ischemia but
distant from detectable histological abnormalities to
establish whether alterations of gap junction organization, in the absence of more gross histological disruption, exist in these regions. A detectable reversible
thallium perfusion defect with associated angina pectoris is suggestive of ischemia affecting the full myocardial
thickness,24-28 but direct demonstration that the epimyocardial samples taken had themselves been subjected to
ischemia was not feasible.
Hypertrophied left ventricle. Left ventricular apical
biopsies were taken during replacement of stenosed
aortic valves in five patients with ECG and echocardiographic evidence of left ventricular hypertrophy.
Processing of Specimens
All biopsies required immediate fixation on removal,
and the specimen was therefore divided as required in
the operating theater, keeping note of tissue orientation
to allow matching of areas analyzed by the different
microscopic techniques described below.
Immunohistochemistry. Tissue was put in Zamboni's
fixative29 (2% paraformaldehyde, 0.2% picric acid, 0.1
mol/L phosphate-buffered saline [PBS], pH 7.4) for 2 to
6 hours. After fixation, all samples were washed in tap
water, dehydrated in alcohol, placed in chloroform, and
embedded in wax according to standard histological
procedures.
The primary antiserum used to localize cardiac gapjunctional protein was raised in rabbits against a synthetic peptide matching residues 131 through 142 of the
cytoplasmically exposed segment of the cardiac gapjunctional protein connexin43.30,31 The peptide, supplied by Dr N.B. Gilula (Research Institute of the
Scripps Clinic, La Jolla, Calif) was prepared by the
simultaneous multiple peptide synthesis procedure of
Houghten32 and was coupled by use of glutaraldehyde to
the carrier protein, keyhole limpet hemocyanin, for
subcutaneous injection into Sandy half-lop rabbits. Full
details of the production and characterization of the
polyclonal antiserum are published elsewhere.30,31,33'34
Sections (10 ,um) of wax-embedded tissue were dewaxed, rehydrated, and incubated in a trypsin solution
(containing 0.1% trypsin [Sigma T-8128], 0.1% CaCl2,
20 mmol/L Trizma base, pH 7.4) for 10 minutes at room
temperature to reexpose antigenic sites masked by
fixation.35 The sections were washed and treated with
0.1 mol/L L-lysine (as blocking agent) in PBS containing
0.1% Triton X-100. Incubation with the primary antiserum (dilution, 1:10 in PBS) was carried out for 1 hour at
37°C. After washing, secondary antibody treatment with
swine anti-rabbit fluorescein isothiocyanate (Dako; 1:20
dilution) was given for 1 hour in the dark at room
temperature. After final washing in PBS, the slides were
mounted with Citifluor mounting medium (Agar Scientific Ltd, Stansted, England). Under the conditions
described, the immunolabeling procedure produces
even and consistent staining through the depth of the
sections.
Immunostained sections were examined by conventional epifluorescence and confocal laser scanning mi-
866
Circulation Vol 88, No 3 September 1993
croscopy. Phase contrast microscopy was used to obtain
details of tissue structure, and adjacent sections stained
with hematoxylin and eosin were routinely examined by
standard light microscopy. Control specimens in which
the antiserum was substituted by preimmune rabbit
serum or buffered saline or the second antibody by
buffered saline were routinely run in parallel. The
specificity of the antiserum for the peptide, for isolated
cardiac gap-junctional protein, and for ultrastructurally
defined gap junctions was demonstrated as part of the
characterization of the antiserum.31,33
For confocal microscopy, immunolabeled sections
were examined with a Bio-Rad Lasersharp MRC-500
running on standard Bio-Rad software.
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Quantitative Analysis
For quantitative assessment of myocardial gap junctions in the normal and diseased groups, all aspects of
tissue processing, labeling, and image acquisition and
analysis were strictly standardized as described below.
Connexin43 gap junction sizes. Measurements of the
longest dimension of the fluorescent spots representing
gap junctions were made by use of the "length" composite command from the digital images of en face
intercalated disks, which are viewed as characteristic
clusters of gap junctions in transversely sectioned tissue.
To confirm that the measurement of the fluorescent
spots by this technique provided a reliable guide to the
true size of gap junctions, despite undulating topology
and different planes of orientation at the ultrastructural
level,36,37 we have compared fluorescent spot sizes with
gap junctions measured on freeze-fracture electron
micrographs of the same tissue and demonstrated a
close correspondence.33'34
Myocardial gap junction surface area. To determine
the total quantity of connexin43 gap junction present in
a volume of myocardium, the tissue section was cut
transverse to the long axis of the constituent myocytes.
The confocal microscope was configured with a small
aperture (aperture control withdrawn 1.2 mm) so that
the confocality of 10 optical slices of data taken at 1-pum
intervals through the depth of the tissue permitted
minimal overlap between consecutive images, ensuring
acquisition of all fluorescence present (Fig 1). These
series of images were collected using the x60 objective
lens and the zoom 1 computer setting (field, 180x 120
1=m2) with a neutral density filter to eliminate significant
fading of fluorescence. The "black level" was constant
(at 4.0) such that the outlines of the individual crosssectioned cells were visible, and the "gain" control was
adjusted so that the spectrum of label intensities
spanned the full 255-level gray scale.
The image data from the volume of tissue of
180x 120x 10 ,um3 were then analyzed with the standard
Bio-Rad software and the more versatile PC IMAGE
(Foster-Findlay Assoc, Newcastle-upon-Tyne, England)
image analysis software. First, the number of crosssectioned myocytes in the fifth optical slice was counted,
and the total area of the field occupied by myocytes was
determined by subtracting from the total sample field all
regions that were either completely devoid of signal
(interstitial fibrosis) or recognizable as blood vessels. A
composite image (or projection) was constructed for
each 10-image series by superimposition (Fig 1), and a
binary overlay was created automatically, in which each
Lam Sure
1pm Inarvas
Optical
Section
Series
1Sqim
FIG 1. Diagrammatic representation of the operation of the
laser scanning confocal microscope. The image of the stimulated fluorescence is acquired through a very small aperture,
resulting in a narrow depth of field, in this case 1 ,m. By
moving the specimen in the z axis (up and down in the
diagram), one can acquire a series of optical sections at set
intervals through the depth of the specimen. Superimposition of
the digitized optical section series produces an in-focus single
image projection of the information in the entire sampled
volume (in this case, 180x120xl0 ,um3). An example of an
optical section series and projection is shown in Fig 4.
pixel was either on or off, as determined by a set
threshold of 60 on the 255 -point gray scale, to eliminate
the background cell outlines. The binary image was then
edited by hand to reject all extraneous lipofuscin and
blood vessel autofluorescence, which were easily identifiable, and an automatic count was made of the total
number of pixels in the on state. This area represented
the total area of labeled gap junction in the sampled
volume of myocytes. Five randomly selected fields of
each tissue sample were analyzed.
To minimize errors introduced by variations between
immunohistochemical labeling runs, samples of each of
the disease groups and a control were included in every
run. The resultant mean binary image area of three
sampling sites from this positive control tissue was used
to ensure consistency of the technique between labeling
runs.
The analysis of the optical section series by this
technique allowed determination of the amount of gap
junction in a defined volume of myocardium expressed
in the standard manner for stereological assessment of
this sort, square micrometers per cubic micrometer.38
In an effort to relate this conventional measure of
surface area of gap junctions per unit volume to the size
of the myocytes composing the tissue from which they
were obtained, a relative index of mean cell volume of
Peters et al Gap Junction Quantification in Human Myocardium
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each of the sampled fields was derived. The volume
index was obtained from the ratio of the number of
cross-sectioned cells to the number of complete disks
present (as an index of cell length) multiplied by the
average cross-sectional area of the myocytes in the
sample volume. If myocytes were cylindrical in shape
with an intercalated disk at each end, the product of cell
length derived by this method and the cross-sectional
area would provide an absolute measure of cell volume.
In reality, myocytes are not simple cylinders; they
possess multiple intercalated disks, all of which lie in the
plane approximately transverse to the long axis of the
cell. Provided that this basic structural arrangement is
present in all groups, however, with equivalent numbers
of intercalated disks per cell, this index of cell volume is
suitable for comparative purposes. The number and
mean cross-sectional area of transected myocytes and
the number of complete or nearly complete (>75%
circumference) collections of gap junctions representing
intercalated disks were determined from the projection
image. The validity of this derivation of an index of cell
volume has been confirmed in an experimental animal
system by comparison with precise values for cell volume determined in isolated myocytes obtained from the
same myocardium (unpublished observations).
Intercalated disks per myocyte: myocardial cell arrangement. To determine the number of intercellular abutments in normal ventricular myocardial architecture
and to detect alterations in disease, counts were made
of the number of cell-surface clusters of gap junction
label, each representing an intercalated disk, in 100
randomly selected isolated left ventricular myocytes
from the normal and ischemic hearts. Myocytes were
isolated by the modified enzymatic tissue dissociation
method,39 as described by Harding et al.40 The cells
produced were immediately fixed and processed for
connexin43 labeling by use of a procedure similar to that
for whole tissue, and confocal microscopy was used to
make disk counts. All focal clusters of label were
considered to represent intercalated disks.
Electron microscopy: gap junction ultrastructure and
connexon packing density. Glutaraldehyde-fixed specimens from the normal and the ischemic hearts were
prepared for thin-section electron microscopy and
freeze-fracture electron microscopy according to standard procedures.41 Glycerinated samples for freezefracture were frozen in Freon, and replicas were preTABLE 1. Details of Normal Patients
Heart
1
Type
WPW
Age
Ventricle
(years)
sampled
37
L
2
Donor
46
L+R
L
3
WPW
28
4
44
L+R
Donor
5
62
L
WPW
Donor
26
R
6
7
37
R
Donor
24
R
Donor
8
WPW, patient with the Wolff-Parkinson-White syndrome; Donor, transplant donor; L, left ventricular sample; R, right ventricular sample.
867
TABLE 2. Details of Patients With Ischemic Heart Disease
Age
Heart
(years)
ECG
9
54
NoQ
10
47
Q
11
46
Q
12
48
Q
13
53
NoQ
Q and No Q, Q waves present/absent in anterior leads on
preoperative ECG. All patients had documented anterior myocardial infarction at least 3 months previously and exercise-induced
ischemia in the anteroapical territory on thallium perfusion scan.
pared in a BaIzers BAF400T apparatus. Replicas and
sections were examined in a Philips EM 301 microscope.
Freeze-fracture electron micrographs (50 pooled
from ischemic and 22 from normal hearts) were used to
measure the connexon surface density within individual
gap junction plaques, since the precise packing arrangement of connexons is reported to alter under hypoxic
conditions (reviewed in References 33 and 37). It was
important for this study that any variation in connexon
density (regardless of the precise arrangement of the
packing) was accounted for in the interpretation of the
measured spot sizes by the immunohistochemical
technique.
An electron micrograph with a final magnification of
x 70 000 of each gap junction plaque was used to
determine the junctional area, using VIDS in software
(Analytical Measuring Systems, Cambridge, England).
The connexon packing density was determined by
counting the number of connexons within a hole in a
mask placed randomly on the micrograph.
Statistical analysis. All results are expressed as
mean±SD. Group data are compared by two-sample t
tests of mean values for each heart. Statistical significance is defined as a value of P<.05.
Results
Details of the 18 patients studied are summarized in
Tables 1 through 3. The ages of the patients from whom
normal left ventricular tissue was obtained were
43.4±12.6 years and those from whom right ventricular
tissue was obtained, 35.4+10.1 years. The five patients
with ischemic heart disease were 49.6±3.7 years old.
The patients with aortic stenosis were 59.6±4.6 years
old.
General Ultrastructure
The ventricular myocardium from the transplant donor and WPW subjects had normal ultrastructural apTABLE 3. Details of Patients With Aortic Valve Stenosis
Valve gradient
Age
Heart
(mm Hg)
(years)
14
60
77
15
56
50
54
16
110
17
63
80
18
65
60
All patients had ECG and echocardiographic evidence of left
ventricular hypertrophy.
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Cfrcnlation Vol 88, No 3 September 1993
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p of left vrmyocanumfrom a patient with Wotff-Parw*lnonWhite syndrome
FI 2. Thin-se n electronmc
showing an electron-dense intexalaed disk (large arrow) traversing
showing normalfeatures. a, Low-power electron mi
between abutting myocyes in the characteristic step-ie manner. The striations of the sarcomeres are aligned in ths healthy tiue
(small arrow). Scale bar=5 ,um. b, Higher-powereectron micrograph showinggap junctions ofnormal appearance (arrows). Note
the characteristic long stretch of gap-junctional embrane at the ends of the disk, consistent with the peripheral ring of large
junctions on immunolabeling (see Fig 3, c). Scale bar=1 ,um
pearances, illustrated in Fig 2. The myocardium sampled from the ischemic hearts had an orderly
arrangement of fibers, with no tissue distortion and no
apparent fibrosis. Various degrees of hypertrophy were
present, but thin-section electron microscopic appearances were otherwise normal. The myocardium from
patients with aortic stenosis had the classic features of
hypertrophy, with myocyte enlargement and hyperchromatic rectangular nuclei, the extent of which varied
from cell to cell.
Immunolabeled Connexin43 Gap Junction
Distribution at Confocal Microscopy
Immunolocalization of connein43 gave consistent
patterns of gap junction staining, common to left and
right ventricle of normal hearts and the left ventricular
myocardium from hearts with aortic stenosis and ischemia. In all these groups, gap junctions were visualized
with the normal adult human patterns previously described42: bright fluorescent domains marking the positions of intercalated disks between myocytes, appearing
as transverse lines of individually resolved spots in
longitudinally sectioned myofibers (Fig 3, a) and discoid
arrays delineated peripherally by large gap junctions in
transversely sectioned myocardium (Figs 3, c and 2, b).
Controls in which antiserum treatment was omitted
or substituted with preimmune serum showed no specific fluorescent labeling (Fig 4, g). Grapelike collec-
tions of lipofuscin (Fig 4, g) with characteristic pink
autofluorescence were present in normal and to a
greater extent in diseased myocardium.
Quantitative Characterization of Gap Junctions
Analsis of connexon density in freeze-fractured gap
junctions. Analysis of data from freeze-fractured gap
junctions .(ig 5) shows that there is no correlation
between gap junction size and the density of its component connexons (r= -.22, P=.13). Fig 6 shows the plot
of these variables for gap junction plaques. of <1 jum2
surface area from the ischemic hearts. There was no
difference in density distribution between myocardium
from the normal and ischemic hearts. Measurement of
total label present in the immunohistochemical images
will therefore be proportional to the number of connexons and the quantity of connexin regardless of any
variation in spot size distribution between different
samples.
Size of immunolabeled gap junctions. Measurement of
the longest axis of the labeled spots of 25 intercalated
disks selected at random from the five normal left
ventricular specimens revealed a mean length of
0.52±0.30 pm, with a distribution as shown in Fig 7, A.
The larger junctions at the periphery of the disk had a
mean length of 0.67+0.32 -m (maximum, 2.8 gum); the
junctions in the central area of the disk, 0.36±0.17 gim
(minimum, 0.1 gum). The distribution of the labeled
Peters et al Gap Junction Quantification in Human Myocardium
869
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FIG 3. Confocal immunolocalization of connexin43 gap junctions in left ventricular myocardium from a patient with
Wolff-Parkinson-White syndrome, showing normal features. a, A single optical section of longitudinally sectioned myocardium
revealing each individual gap junction domain as a separate spot (arrow) grouped within intercalated disks. Scale bar=50 gtm. b,
A projected optical series from transversely sectioned myocardium showing the characteristic features of a disk viewed en face
(arrow), with a peripheral ring of large junctions and smaller central domains. Scale bar=50 ,gm. c, High-power view of en face
disks, showing the entire gap junction population of each disk. Scale bar=-0 gm.
870
Circulation Vol 88, No 3 September 1993
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FIG 4. A confocal optical section series of l-um intervals through the depth
of transversely sectioned normal human myocardium labeled for connexin43
(compare with Fig 1). Scale bars=50 gm. a through e, The first, third, fifth,
seventh, and ninth optical sections in an optical series acquired for the
quantification procedure. There are progressive changes in the image content
throughout the series. f Theprojection of the 10 images of the optical section
series (a through e), showing some completely defined intercalated disks with
their entire gap junction population displayed in focus. g, A wax section
adjacent to that in a through f in which preimmune serum was substituted
for the HJ antiserum in the processing procedure. This control section
contains no fluorescent label, demonstrating the specificity of the procedure,
and shows the lipofuscin autofluorescence, which can readily be distinguished from labeling (when present) for the purposes of editing the image.
8 '71
Peters et al Gap Junction Quantification in Human Myocardium
A - from normal left ventricle
25
bC 20-
10
z5
0
Measured Spot Size (pm)
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B
-
from ischemic left ventricle
25
b
20-
-a
0
15
'a
FIG 5. Electron micrograph offreeze-fracture replica showing the densely packed connexon particles (arrow) of an entire
junctional plaque (gj) from a patient with ischemic heart
disease. Scale bar=0.5 gum.
junction long-axis measurements pooled from the ischemic left ventricles (mean, 0.54±0.42 gm) showed no
difference from normal (Fig 7, B).
Gap-junctional surface area per unit volume of tissue.
An example of images from an optical section series of
C-M 150E
0
125
-
100
-
a-
=
L
o
. .-.
o
75-
X
0'50
.9
0.00
0.25
.50
.75
1.00
gap junction plaque area (pm2)
FIG 6. Plot of connexon surface density vs gap junction
plaque area in plaques up to 1 gm2 pooled from ventricular
myocardium from the ischemic hearts. There is no correlation
(P=.18), and the slope is not significant.
00
5
IN.
0
0
0
000
0
w
v_-
Pm
"
W _
.
-
M
-
m
m
N
W W
Measured Spot Size (pm)
FIG 7. Frequency distributions of long-axis measurements of
labeled spots in confocal images (A) from the normal left
ventricles (mean, 0.52 gum) and (B) from the ischemic left
ventricles (mean, 0.54 gum). There is no difference between
these distributions.
connexin43-labeled transversely sectioned ventricular
myocardium prepared for quantitative analysis is shown
in Fig 4. The results of the morphometric analysis of
such images to determine myocardial gap junction content in the healthy and diseased hearts are shown in
Table 4. Normal adult left ventricular myocardium had
a gap junction surface area of 0.0051±0.0015 gm2/,um3
myocyte volume, and normal right ventricle showed no
significant difference (0.0044±0.0006 ,um2Im3). Left
ventricular myocardium from recurrently ischemic
hearts had a significantly reduced gap junction surface
area of 0.0027±0.0009 gm2/,um3 (P=.024). Hypertrophied left ventricle from patients with aortic stenosis
also had a reduced gap junction surface area of
0.0031±0.0005 11m2/gm3 (P=.05) and was not significantly different from myocardium from the ischemic
hearts.
872
Circulation Vol 88, No 3 September 1993
TABLE 4. Results of Quantitative Morphometric Analysis
Gap junction surface
area/volume myocardium
Heart
1
2
3
4
S
2
4
6
7
8
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9
10
11
12
13
(gm2/Im3)
Cell volume
index
0.00476±0.00076
0.00751±0.00158
0.00419±0.00072
0.00360±0.00117
0.00540±0.00113
29 436±5377
15 347±859
23 516±5534
24 668±6998
20 925±1170
Mean LV
0.00508±0.00151
22 778±5174
RV
RV
RV
RV
RV
0.00501±0.00106
0.00349±0.00034
0.00464±0.00123
0.00402±0.00038
0.00473±0.00050
22 799±3512
22 582±3136
26 901±8047
22 577±3146
22 639±3424
Mean RV
0.00438±0.00061
23 499±1904
ILV
ILV
ILV
ILV
ILV
0.00230±0.00098
0.00199±0.00038
0.00193±0.00046
0.00351±0.00162
0.00388±0.00099
39 067± 1049
36 756±9360
29 346±6100
25 723±6528
24 443±3072
0.00272±0.00091*
31 067±6553
0.00352±0.00029
0.00265±0.00033
0.00333±0.00021
0.00253±0.00080
0.00359±0.00118
29 070±3853
32 804±4312
34 204±4156
35 104±1179
26 052±6612
0.00313±0.00050*
31 447±3794*
Tissue type
LV
LV
LV
LV
LV
Mean ILV
14
15
16
17
18
HLV
HLV
HLV
HLV
HLV
Mean HLV
Mean, mean ± SD of means of five sample fields for each heart. Values are mean±SD. LV, normal left
ventricle; RV, normal right ventricle; ILV, ischemic left ventricle; HLV, hypertrophied left ventricle.
*Significantly different from value for normal left ventricle (P<.05).
Comparative index of myocyte volume. The cell volume
index derived from the hearts with aortic stenosis
demonstrated the increased ventricular myocyte volume
that would be expected in hypertrophied myocardium
(31 477± 3794 units) compared with normal (22 778+
5174 units; P=.019). The mean value for myocardium
from the ischemic ventricles (31 067±6553 units), although increased, did not differ significantly from normal (P=.062), owing to the presence of a wider range of
cell sizes than the aortic stenosis group.
Since the cell volume indexes were derived from the
same tissue fields as the gap-junctional surface density
values, these data allow approximation of an index of
gap junction area per myocyte for each myocardial
specimen, derived from the mean of the gap junction
areas and the cell volume indexes of each of the five
sample volumes. This value is reduced in myocardium
from the ischemic hearts (10 758±2133 units) compared
with normal (14 996±2268 units; P=.019). Hypertrophied myocardium, by comparison, has a value of
12 848±1533 units (P=.11). These results and their
interpretation must be treated with caution because the
ratio used in their derivation may artificially exaggerate
differences.
Intercalated disk counts. A confocal image of a labeled
isolated ventricular myocyte is shown in Fig 8. When
such images were used to count the number of surface
clusters of connexin43 label (each representing an intercalated disk) per myocyte, there was no difference
between cells from myocardium of normal (11.55±2.20)
and ischemic hearts (11.87+2.49; P=.33).
Discussion
Quantitative studies of gap junctions in mammalian
myocardium, most of which used thin-section electron
microscopy, have been reviewed by Page.4 These studies
are confined to species other than humans, in whom
characterization of myocardial gap junctions and alterations that may accompany human myocardial disease
have therefore not been determined. We have published findings at the borders of healed human infarcts22
Peters et al Gap Junction Quantification in Human Myocardium
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FIG 8. Confocal microscopic projection image of an optical
section series through a single isolated ventricular myocyte
labeled for connexin43, showing the positions of the intercalated disks as clusters of transversely oriented labeled gap
junctions (examples arrowed). Scale bars=20 ,um.
but have not previously attempted quantification of gap
junction content.
The group of ischemic patients tended to be older
than the group with normal left ventricles (49.6±3.7 vs
43.4±12.6 years), a difference that is difficult to overcome given the reasons for which these groups undergo
surgery. However, this difference was not statistically
significant (P<.35), and there were no trends to suggest
age-related changes in any parameters within any of the
study groups.
The gap-junctional surface area in normal human left
ventricular myocardium (0.0051 ,um2/pum3 myocyte volume), as determined in the present study, falls within
the range of values reported from electron microscopic
ultrastructural analyses of myocardium of other mammalian species. These include rabbit ventricle (0.017
I£m2/j&m3),43 rat ventricle (0.0047 and 0.0022 to 0.0154
gm2/1,m3),44,4 and canine ventricle (0.0085 11m2/4m3),46
from which Luke et a138 have also estimated a value of
0.0052 Mm2/1m3 by an immunohistochemical technique
using light microscopy. In contrast with all these reports,
the technique used in the present study enables the
quantity of gap junction to be related directly to entire
and completely visualized intercalated disks sampled
through relatively large volumes of tissue. The quantitative spatial distribution data obtained facilitate meaningful comparisons between disease groups and could,
in principle, allow direct correlation with myocardial
function and electrophysiological data acquired before
myocardial fixation.
The presence of fluorescent labeling must be interpreted with some caution, since the antibody is directed
to a small segment of the connexin43 molecule and
labeling may not necessarily represent intact or functional gap junctions. However, the agreement of our
data with the values obtained by standard electron
microscopic morphometry,43-46 the correlation of the
labeling patterns with gap junction distribution deter-
873
mined by ultrastructural techniques,22 and the confirmation by immunogold labeling at the electron microscopic level of specific binding of the antibodies to gap
junctions,33 all serve to support the validity of the
approach.
Our finding of a mean of 11.55 +2.2 intercalated disks
per myocyte in normal human epimyocardium is consistent with data reported by Luke and Saffitz,47 who used
a quantitative electron microscopic technique to determine a mean of 11.2 intercalated disk contacts per
myocyte in normal canine ventricular myocardium.
These authors showed a reduced number (6.5) of such
intercellular contacts in canine myocardium associated
with infarction but had specifically selected regions
associated with fibrotic scarring. In the present study,
however, we obtained normal intercalated disk counts
in ischemic hearts, despite changes in cell size distribution, suggesting that the basic architecture of intercellular abutments was not significantly altered in regions
free of scar.
Substrate for Abnormal Impulse Propagation
It has long been suggested that fibrosis plays a part in
the anatomy of arrhythmogenesis,48 but the results of
the present study suggest a mechanism by which intercellular coupling may be impaired in the ischemic heart
without invoking fibrosis as the explanation.
Both ischemic heart disease and left ventricular hypertrophy are associated with ventricular arrhythmias
and sudden cardiac death.49,50 The development of
treatment strategies for cardiac arrhythmias that occur
in these and other clinical settings requires a detailed
and complete understanding of the mechanisms by
which these arrhythmias are generated. One of the
many adverse effects of cardiac ischemia is a decrease in
conduction velocity,12'51 a factor increasing the likelihood of arrhythmias. Although gap junctions are established as the organelle determining intercellular and
whole-tissue conductance and conduction velocity4J12'51
and an absence of gap junctions results in electrical
isolation,52 the relation between the relative abundance
of gap junctions, intercellular conductance, and conduction velocity is poorly understood. Evidence suggests
that regions of the heart with slow conduction and
relatively low intercellular conductance (such as the
atrioventricular node) contain fewer gap junctions than
regions of more rapid conduction.5' The possibility
exists, therefore, that a reduction in gap junction surface area per unit volume of about 40% in ventricular
myocytes from ischemic and hypertrophied ventricles,
as demonstrated in the present study, may significantly
alter patterns and rates of impulse propagation. The
distribution of labeled gap junction long-axis lengths
and the number of intercalated disks per cell remained
unchanged in the myocardium from the ischemic hearts
compared with normal, suggesting that the reduction in
gap-junctional content occurs as a result of reduction in
the numbers of all sizes of junction. This is of interest
because electron microscopic examination of the canine
infarct border region has suggested a preferential reduction of larger junctions, but fibrotic scarring of the
canine myocardium may be relevant to this difference.47
The greater myocyte volume that characterizes hypertrophied myocardium associated with chronic aortic
stenosis was evident in the present study. Compensatory
874
Circulation Vol 88, No 3 September 1993
hypertrophy that might be expected in the myocytes of
the myocardium around a healed infarct23,53 did not
occur to a statistically significant degree, despite a
greater mean cell volume, owing to the large standard
deviation.
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Functional Significance: Correlating Morphology
With Electrophysiology
Although electrophysiological studies of arrhythmogenic cardiac disease have documented significant
abnormalities of active membrane properties,8 many
have failed to do So.13'14 Boyden et al14 concluded that
alteration of unspecified morphologic features of the
myocardium was responsible for the electrophysiological disturbances in chronically hypertrophied canine
atria. Kieval et allS demonstrated normal action potential characteristics but abnormal gap-junctional intercellular conductance in postischemic myocyte pairs, and
reduced junctional conductance in cultured myocytes
infected with Trypanosoma cruzi is associated with a
qualitative reduction in connexin43 expression.54 A reduction in gap-junctional coupling between myocytes
may, therefore, be an important morphological feature
that could interact with altered active membrane properties in diseased myocardium of a variety of pathogeneses. It is likely that arrhythmogenic states result from a
combination of changes of both membrane properties
and coupling characteristics of the myocardium, and it
has been suggested that abnormal cellular coupling may
be the predominant electrophysiological derangement
at the border of a healed myocardial infarct.55
It has not been possible to study the interaction
between the action potential and myocardial resistivity
during programmed stimulation,55-58 but a number of
computer models simulating this interaction have been
developed. Joyner59 showed that as coupling resistance
increases, the "intrinsic" cellular differences of action
potential duration become increasingly manifest, and
Lesh et al55 showed a coincident increase in axial
resistivity, leading to slowed conduction. The combination of dispersion of action potential duration and slow
conduction promotes reentrant tachycardia initiation
and perpetuation,12 and Quan and Rudy's60 model
suggests that cellular uncoupling is arrhythmogenic by
just these mechanisms.
Implications of Results
A reduction in gap junction coupling may, therefore,
change conduction velocity and unmask intrinsic differences in action potential characteristics, manifest at the
clinical level by nonspecific changes in the QRS complex
on the ECG, changes in the coordination of contraction
and the mechanical efficiency of the heart, and a
possible lowering of arrhythmic threshold. These phenomena are well recognized in ischemic and hypertrophied hearts. The process of uncoupling may, itself,
induce changes in action potential characteristics55'61
and recovery of excitability.55 All these alterations
increase the potential for spontaneous arrhythmias,62
for which there is growing evidence of the role of
abnormal intercellular coupling.1"2'55'6' The results of
the present study suggest that changes in resistivity, as
evidenced by altered gap junction quantity in the diseased human ventricle, may indeed occur.
The resistivity of myocardium,
a potentially imporarrhythmias, has been ignored in
many studies confined to relating active membrane
properties to the behavior of the tissue and the heart as
tant factor in clinical
a whole. In the present study, normal human ventricular
myocardium has been characterized with respect to gap
junctions. Abnormalities of gap junction content, as
demonstrated in ischemic and hypertrophied hearts,
would be consistent with the clinically apparent disturbances of ventricular electromechanical function in
these conditions, the explanation and treatment of
which remain poorly understood. Little is known of the
action of conventional antiarrhythmic agents on cellular
coupling and anisotropy,63 and with a number of clinical
trials questioning the conventional wisdom of pharmacological arrhythmia treatment,64 a broader approach to
understanding electromechanical dysfunction may require greater attention to gap junction intercellular
coupling.
Acknowledgments
This study was supported by the British Heart Foundation
(grant F268). We are grateful to John Pepper, Graeme Bennett, and Sir Magdi Yacoub for providing the surgical specimens. The photographic expertise and assistance of Stephen
Rothery is gratefully acknowledged. We thank Federica Del
Monte for the myocyte isolation.
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N S Peters, C R Green, P A Poole-Wilson and N J Severs
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Circulation. 1993;88:864-875
doi: 10.1161/01.CIR.88.3.864
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