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JACC Vol. 32, No. 3
September 1998:800 –7
Effects of Angiotensin II on Expression of the Gap Junction Channel
Protein Connexin43 in Neonatal Rat Ventricular Myocytes
STEPHEN M. DODGE, MD,* MICHAEL A. BEARDSLEE, MD, BRUCE J. DARROW, MD, PHD,†
KAREN G. GREEN, BA, ERIC C. BEYER, MD, PHD,‡ JEFFREY E. SAFFITZ, MD, PHD, FACC
St. Louis, Missouri
Objectives. To elucidate signal transduction pathways regulating expression of myocardial gap junction channel proteins (connexins) and to determine whether mediators of cardiac hypertrophy might promote remodeling of gap junctions, we characterized
the effects of angiotensin II on expression of the major cardiac gap
junction protein connexin43 (Cx43) in cultured neonatal rat
ventricular myocytes.
Background. Remodeling of the distribution of myocardial gap
junctions appears to be an important feature of anatomic substrates of ventricular arrhythmias in patients with heart disease.
Remodeling of intercellular connections may be initiated by
changes in connexin expression caused by chemical mediators of
the hypertrophic response.
Methods. Cultures were exposed to 0.1 mmol/liter angiotensin
II for 6 or 24 h, and Cx43 expression was characterized by
immunoblotting, confocal microscopy and electron microscopy.
Results. Immunoblot analysis revealed a twofold increase in
Cx43 content in cells treated for 24 h with angiotensin II (n 5 4,
p < 0.05). This response was inhibited by the presence of 1.0
mmol/liter losartan, an AT1-receptor blocker. Confocal and electron microscopy demonstrated enhanced Cx43 immunoreactivity
and increases in the number and size of gap junction profiles in
cells exposed to angiotensin II for 24 h. These effects were also
blocked by losartan. Immunoprecipitation of Cx43 from cells
metabolically labeled with [35S]methionine demonstrated 2.4- and
2.9-fold increases in Cx43 radioactivity after 6 and 24 h exposure
to angiotensin II, respectively (p < 0.03 at each time point).
Conclusions. Angiotensin II up-regulates gap junctions in
cultured neonatal rat ventricular myocytes by increasing Cx43
synthesis. Signal transduction pathways activated by angiotensin
II under pathophysiologic conditions could initiate remodeling of
conduction pathways, leading to the development of anatomic
substrates of arrhythmias.
(J Am Coll Cardiol 1998;32:800 –7)
©1998 by the American College of Cardiology
Reentrant ventricular tachycardias in patients with healed
myocardial infarcts depend on regions of slow heterogeneous
conduction and unidirectional conduction block that typically
map to viable but structurally altered myocardium bordering
the healed infarct scar (1– 4). Although conduction through
these regions is highly abnormal, intracellular action potential
recordings in infarct border zone myocytes are typically normal
or nearly normal (3,4). These observations indicate that derangements in conduction critical in reentrant arrhythmogenesis arise not primarily from abnormalities of active sarcolemmal ionic currents but rather from alterations in intercellular
current transfer. This conclusion is supported by results of
morphometric and immunofluorescence studies that have revealed extensive remodeling of the spatial distribution of gap
junctions in myocardium bordering infarct scars (2,5–7). During the process of infarct healing, not only is necrotic muscle
replaced by scar tissue, but viable myocardium at the edges of
the infarct develops interstitial fibrosis characterized by accumulation of collagen bundles oriented parallel to the long axis
of the myocytes (8,9). The normal network of extensive
intercellular connections between neighboring myocytes in
end-to-end and side-to-side apposition becomes remodeled
such that there is a substantial loss of gap junctions between
cells in side-to-side orientation (5,6). This rearrangement of
connections impairs transverse propagation and creates regions of slow heterogeneous conduction and unidirectional
conduction block (2,6).
Although peri-infarct myocytes eventually become interconnected by fewer and smaller gap junctions than normal cells
once infarcts become fully healed, the initial events in this
remodeling process probably involve dynamic changes in gap
junction protein (connexin) expression and distribution. This
suggestion is supported by the results of studies showing that
during the active inflammatory phase of infarct healing, viable
myocytes at the edge of the infarct lose the normal pattern of
From the Departments of Pathology, Medicine and Pediatrics, Washington
University School of Medicine, Saint Louis, Missouri. This study was supported
by National Institutes of Health Grants HL50598 and HL45466.
Manuscript received September 2, 1997; revised manuscript received May 4,
1998, accepted May 15, 1998.
Address for correspondence: Dr. Jeffrey E. Saffitz, Department of Pathology,
Box 8118, Washington University School of Medicine, 660 S. Euclid Avenue,
Saint Louis, Missouri 63110. E-mail: [email protected].
*Present address: Department of Internal Medicine, University of Minnesota School of Medicine, Minneapolis, Minnesota 55410.
†Present address: Department of Internal Medicine, Columbia-Presbyterian
Medical Center, New York, New York 10032.
‡Present address: Section of Pediatric Hematology/Oncology, University of
Chicago Children’s Hospital, Chicago, Illinois 60637.
©1998 by the American College of Cardiology
Published by Elsevier Science Inc.
0735-1097/98/$19.00
PII S0735-1097(98)00317-9
JACC Vol. 32, No. 3
September 1998:800 –7
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EFFECTS OF ANGIOTENSIN ON Cx43 EXPRESSION
801
Materials and Methods
Abbreviations and Acronyms
cAMP
5 cyclic adenosine 39-59 monophosphate
Cx43
5 connexin43
IgG
5 immunoglobulin G
PBS
5 phosphate-buffered saline
SDS–PAGE 5 sodium dodecyl phosphate polyacrylamide gel
electrophoresis
TPBS
5 phosphate-buffered saline containing 0.5% Triton X-100
large gap junctions concentrated at ends of myocytes and
exhibit a distinctly different pattern in which many small
junctions become distributed uniformly over the cell surface
(2,7). It is unknown whether connexin expression is increased
in these myocytes during early infarct healing, but the marked
rearrangement in gap junction distribution suggests that significant changes in connexin synthesis, assembly into channels
and degradation may accompany the structural changes taking
place in gap junctions. It seems likely that during this phase of
infarct healing multiple signal transduction pathways are activated in both myocytes and nonmyocytic cells in the heart in
response to cytokines and neurohumoral factors. These complex signaling events probably stimulate expression of multiple
genes and alter protein synthesis and degradation dynamics, all
of which eventually culminate in a chronic, anatomically stable
state of tissue remodeling.
Among the potential mediators implicated in ventricular
remodeling after myocardial infarction is angiotensin II
(10,11). Results of multiple clinical trials have shown that
administration of angiotensin-converting enzyme inhibitors to
patients after myocardial infarction improves cardiac function,
prolongs survival and modulates ventricular remodeling (12–
17). The specific mechanisms responsible may be multiple and
probably involve both direct effects on the renin-angiotensin
system and inhibition of kinin degradation. Angiotensin II has
also been shown to elicit a hypertrophic response in cultured
neonatal rat cardiac myocytes involving activation of many
cardiac genes (18 –23).
Work in our laboratory has focused on the hypothesis that
during the development of anatomic substrates of arrhythmias,
chemical mediators of the hypertrophic response activate
synthesis of gap junction channel proteins, which we propose is
an obligatory first step in the remodeling of sites of intercellular coupling. In previous studies (24), we showed that
long-term exposure (up to 24 h) of cultured neonatal rat
ventricular myocytes to the membrane-permeable cyclic adenosine 39-59 monophosphate (cAMP) analog dibutyryl cAMP
increased expression of the ventricular gap junction channel
protein connexin43 (Cx43) and also increased the speed of
impulse propagation. To characterize further the potential role
of mediators of hypertrophy in the development of anatomic
substrates of arrhythmias, we performed the present studies to
determine whether long-term exposure (24 h) of cardiac
myocytes to angiotensin II affects expression of Cx43.
Isolation and Culture of Neonatal Rat Ventricular
Myocytes
Primary cultures of 1- to 2-day old neonatal rat ventricular
myocytes were prepared as reported previously (24,25).
Briefly, hearts were trimmed of atrial tissue and great vessels,
minced on ice and dissociated with collagenase. Cells were
resuspended in PC-1 medium (Hycor Biochemical, Irvine,
California) supplemented with 100 U/ml penicillin, 100 mg/ml
streptomycin, 250 ng/ml amphotericin and 0.1 mmol/liter bromodeoxyuridine, and seeded onto 60-mm polystyrene culture
dishes without preplating at a density of 1.5 3 105 cells/cm2.
Because this medium is serum-free, it contained no exogenous
source of angiotensin, angiotensinogen or other growth factors. Culture medium was replenished daily. Cultures were
maintained for 72 h before being treated for selected intervals
with 0.1 mmol/liter angiotensin II and analyzed for the effects
of connexin expression. In some experiments, the effects of 0.1
mmol/liter angiotensin II and 1.0 mmol/liter dibutyryl cAMP
were compared by analyzing cultures exposed to either mediator.
Immunoblotting of Cx43. Multiple myocyte cultures were
treated with angiotensin II or dibutyryl cAMP for 6 or 24 h.
After exposure to mediators, cells were rinsed with phosphatebuffered saline (PBS), scraped into PBS containing 10 mg/
100 ml aprotinin and lysed with sonication on ice with 4 bursts
of 15 s each. Equal volumes of protein extracts from each dish
were solubilized and electrophoresed on 12.5% polyacrylamide gels. Proteins were transferred to Immobilon-P (Millipore, Bedford, Massachusetts) using a semidry transfer apparatus (BioRad, Hercules, California) at 3 W for 1 h. The
membranes were blocked overnight in 2% gelatin in PBS and
incubated in a mouse monoclonal anti-Cx43 antibody (MAB
3068, Chemicon International, Temecula, California) at a
dilution of 1:1,000 in phosphate-buffered saline containing
0.5% Triton X-100 (TPBS). After five washes in TPBS, the
blots were incubated with horseradish peroxidase-conjugated
goat antimouse immunoglobulin G (IgG) (Boehringer Mannheim, Indianapolis, Indiana) which was diluted 1:2,000 in
TPBS. After being rinsed 6 times for 5 min each in TPBS, the
blots were incubated for 1 min in ECL solution (Amersham,
Arlington Heights, Illinois) and exposed to x-ray film (Kodak,
New Haven, Connecticut). Densitometric quantification of
signal intensity was performed as previously described (26).
Background measurements of signal intensity were subtracted
individually from each lane.
Immunohistochemistry and confocal microscopy. Myocytes on glass coverslips were rinsed with PBS, fixed for 20 min
in 4% paraformaldehyde in PBS at room temperature and
permeabilized and blocked with 0.3% Triton X-100/3% normal
goat serum/1% bovine serum albumin in PBS for 10 min at
room temperature. Cells were incubated with mouse monoclonal anti-Cx43 antibody (MAB 3068, Chemicon International)
overnight at 4°C, washed extensively and then incubated with
CY3-conjugated goat antimouse IgG (Jackson Immunolabs,
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DODGE ET AL.
EFFECTS OF ANGIOTENSIN ON Cx43 EXPRESSION
West Grove, Pennsylvania) for 3 h. Quantification of immunofluorescent labeling was performed using a Molecular Dynamics Sarastro 2000 laser scanning confocal microscope
(Sunnyvale, California). Random high-power fields of myocytes were selected for image analysis under phase-contrast
viewing conditions. The number of pixels occupied by the high
intensity immunofluorescent signal in discrete regions of intercellular apposition was automatically counted in each field
using the image analysis software. The high intensity of this
signal, which appeared to localize exclusively to gap junctions,
was likely due to the extremely dense packing of connexin
molecules in gap junction channel arrays. The image analysis
protocol eliminated lower intensity immunofluorescent signal
within cells that probably included both specific Cx43 signals in
intracellular sites and nonspecific signals. Confocal data were
expressed as the number of pixels that exhibited strong immunofluorescent signals divided by the total number of pixels
occupied by cells.
Electron microscopy. Angiotensin II-treated and control
cultures were fixed and prepared for electron microscopy as
previously described (5). Intercalated disk and gap junction
profile lengths and total sarcolemma lengths and myocyte
areas were measured in randomly photographed groups of
interconnected myocytes to determine the relative number and
size of gap junctions per unit intercalated disk length, unit
sarcolemma length and unit cell area, as previously described
(5).
Metabolic labeling and immunoprecipitation assays. Cultures were treated for 6 or 24 h with angiotensin II or dibutyryl
cAMP and, during the final 2 h of the treatment interval, were
also incubated in methionine-depleted medium containing
[35S]methionine (100 mCi/ml, Amersham, Arlington Heights,
Illinois), as previously described (24,25). At the completion of
the treatment interval, cells were washed to remove unincorporated radioactivity. Protein extracts were prepared as previously described and incubated with 20 ml antimouse IgGconjugated Sepharose D beads (Pierce, Rockford, Illinois) and
3 ml anti-Cx43 mouse monoclonal antibody for 2 h at 4°C with
agitation. The beads were collected by centrifugation, washed
overnight and then washed three times for 30 min in radioimmunoprecipitation assay buffer (0.6% sodium dodecyl sulfate
[SDS], 1% Triton X-100 in PBS with aprotinin, Pefabloc
[Boehringer Mannheim, Indianapolis, Indiana], sodium orthovanadate and sodium fluoride, 10 mg/100 ml each) at 4°C.
Radiolabeled proteins were separated by sodium dodecyl
phosphate polyacrylamide gel electrophoresis (SDS–PAGE)
on 12.5% gels and analyzed by fluorography after treatment
with ENH3ANCE (New England Nuclear).
Statistical Analysis
Results are reported as mean 6 SD. Differences between
groups were considered significant at p , 0.05. Control and
test values in cultures treated with angiotensin II for selected
intervals were compared by one way analysis of variance with
Fisher’s protected least significant difference post hoc testing
JACC Vol. 32, No. 3
September 1998:800 –7
Figure 1. A, Representative immunoblot comparing the amount of
Cx43 in control cultures and cultures treated for 6 or 24 h with
angiotensin II (A-II) or for 24 h with dibutyryl cAMP (db-cAMP).
Hatch marks to the right of the numbers in A indicate the positions of
molecular weight markers from 66 to 14 kDa. B, Densitometric
quantification of immunoblot band intensities in cultures treated with
angiotensin (A-II) for 6 or 24 h or dibutyryl cAMP (db-cAMP) for 6 h.
The angiotensin effect was blocked by losartan (Los). The values
(means 6 SD) from treated cultures have been normalized to values in
control cultures in each of four separate experiments. Asterisks
indicate p , 0.05 compared with controls.
using a statistical analysis software package (Statview, Abbacus
Concepts, Inc, Berkeley, California). Electron microscopic
data were compared with unpaired t tests.
Results
Effects of angiotensin II on Cx43 content in cultured
myocytes. To determine whether the total amount of Cx43
protein changed in cultures exposed to angiotensin II, total
protein from control and treated cultures was separated by
SDS–PAGE and analyzed by immunoblotting. Figure 1 shows
a representative immunoblot and quantitative results from
four separate experiments in which Cx43 band intensities were
JACC Vol. 32, No. 3
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EFFECTS OF ANGIOTENSIN ON Cx43 EXPRESSION
803
Figure 2. Representative confocal microscopic images of
Cx43 immunoreactive signal in control myocytes (left panel)
and myocytes exposed to angiotensin II for 24 h (right
panel). The gap junctional signal appears as bright, punctate
spots concentrated at sites of cellular apposition. A greater
number and larger size of spots is apparent in the treated
cultures.
analyzed densitometrically and normalized to the value measured in untreated, control cultures. Cx43 migrated as two
major bands at approximately 46 and 43 kDa. These bands
correspond to the positions of phosphorylated isoforms of
Cx43 (27), which comprises the majority of Cx43 in cardiac
myocytes (28). Exposure of myocytes to 0.1 mmol/liter angiotensin for 6 h resulted in a modest (,1.5-fold) increase in Cx43
content that did not achieve statistical significance. However,
exposure of myocytes to angiotensin for 24 h resulted in a
twofold increase in Cx43 content (p , 0.05). The increase in
Cx43 content after 24 h of angiotensin exposure was blocked by
losartan.
Effects of angiotensin II on gap junctions in cultured
myocytes. To determine whether the increased content of
Cx43 in cells treated with angiotensin II was associated with
changes in gap junctions, we performed immunofluorescence
studies. Control cultures and cultures treated with 0.1 mmol/
liter angiotensin II for 24 h were incubated with monoclonal
antibodies against Cx43 and analyzed by quantitative laser
scanning confocal microscopy. Figure 2 shows representative
confocal images and Figure 3 shows the results of quantitative
digital image analysis of immunostained control and angiotensin II-treated myocytes in four separate experiments. Treatment of myocytes with angiotensin II for 24 h caused an
increase in Cx43 immunoreactive signal compared with untreated control myocytes as judged by inspection of confocal
images (Fig. 2). Quantitative analysis revealed a twofold
increase in the area occupied by Cx43 immunoreactive signals
(Fig. 3), consistent with the results of immunoblotting shown in
Figure 1. The increase in the immunoreactive signal area was
blocked completely when cultures were incubated with angiotensin in the presence of losartan.
Additional angiotensin-treated and control cultures were
examined by electron microscopy. Figure 4 shows representative electron microscopic images of intercalated disk regions
connecting control myocytes and myocytes treated for 24 h
with angiotensin II, and Table 1 shows morphometric data
from two sets of treated and control cultures. A total of 80
myocytes in 13 fields from angiotensin-treated cultures and 66
myocytes in 8 fields from control cultures were analyzed. Both
the number and size of individual gap junction profiles were
increased in cells treated with angiotensin II for 24 h. There
was no apparent increase in the total length of intercalated
disk profiles per unit myocyte area. These data imply that
angiotensin II had a relatively selective effect on gap junctions
without affecting other junctional components. However, we
did not perform morphometric analyses of other organelles
such as sarcomeres, mitochondria or components of the sarcoplasmic reticulum. The changes in gap junctions may be only
Figure 3. Quantitative confocal analysis of Cx43 immunofluorescence
signal in cultures treated for 24 h with angiotensin II (A-II) in the
presence or absence of losartan (Los). Values (means 6 SD) have
been normalized to values in control cultures in each of four separate
experiments. Asterisks indicate p , 0.02 compared with controls.
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DODGE ET AL.
EFFECTS OF ANGIOTENSIN ON Cx43 EXPRESSION
JACC Vol. 32, No. 3
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Figure 4. Representative electron micrographs of sites
of intercellular connections in control myocytes (A)
and myocytes exposed to angiotensin II for 24 h (B).
Individual gap junction profiles are identified by arrows. Bar 5 1.0 mm.
part of a more generalized hypertrophic growth response
induced by angiotensin II.
Effects of angiotensin II on Cx43 synthesis. To identify
potential mechanisms responsible for the increase in Cx43
content caused by angiotensin II, we performed metabolic
labeling studies to compare the rates of Cx43 synthesis in
Table 1. Ultrastructural Morphometric Analysis of Gap Junctions
Number of GJ profiles per
100 mm ID length
Aggregate GJ profile length (mm)
per 100 mm ID length
Aggregate GJ profile length (mm)
per 1,000 mm2 myocyte area
Aggregate ID length (mm) per
1,000 mm2 myocyte area
Individual GJ profile length (mm)
Angiotensin II
Control
29.5 6 15.2*
7.6 6 8.1
8.6 6 5.8*
1.5 6 1.7
2.6 6 2.1*
0.49 6 0.6
28.1 6 4.7
27.9 6 16.6
0.31 6 0.11*
0.21 6 0.07
*p , 0.02. GJ 5 gap junction; ID 5 intercalated disk.
control cells and treated cells. Cells were exposed to angiotensin II or to dibutyryl cAMP for 6 or 24 h. During the final 2 h
of each exposure interval, the cells were incubated with
[35S]methionine. The cells were lysed and the amount of
radioactivity incorporated into Cx43 was measured with quantitative immunoprecipitation assays. Because the half-life of
Cx43 in cultured neonatal rat ventricular myocytes is approximately 3 h (25,29), the amount of radioactivity incorporated
into Cx43 during a 2-h labeling interval was assumed to be
determined mainly by the rate of Cx43 synthesis.
Figure 5 shows a representative immunoprecipitation assay
and quantitative results from four separate metabolic labeling
and immunoprecipitation experiments. The amount of radiolabeled Cx43 increased by 2.4- and 2.9-fold in cells treated with
angiotensin II for 6 or 24 h, respectively (p , 0.03 for each
compared to controls). This effect was blocked by losartan.
These results indicate that although the total content of Cx43
had not increased by a significant amount after a 6-h exposure
to angiotensin II, the rate of incorporation of [35S]methionine
JACC Vol. 32, No. 3
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DODGE ET AL.
EFFECTS OF ANGIOTENSIN ON Cx43 EXPRESSION
Figure 5. A, Representative immunoprecipitation assay showing the
amount of radioactivity incorporated into Cx43 during a 2-h interval of
metabolic labeling in control cultures or in cultures treated with
angiotensin II (A-II) for 6 or 24 h, angiotensin plus losartan (A-II 1
Los) for 24 h, or dibutyryl cAMP (db-cAMP) for 6 or 24 h. B,
Densitometric quantification of signal intensity in immunoprecipitation studies of control cultures or cultures treated with angiotensin II
(A-II) (n 5 4 for each), angiotensin II plus losartan (A-II 1 Los) (n 5
4) or dibutyryl cAMP (db-cAMP) (n 5 2). Values (means 6 SD) have
been normalized to control values in each experiment. Asterisks
indicate p , 0.03.
into Cx43 had increased significantly during the final 2 h of a
6-h exposure to angiotensin. The increased rate of incorporation persisted such that it was nearly threefold greater than
control values during the final 2 h of a 24-h exposure to
angiotensin II, by which time the total content of Cx43 had
doubled. In contrast, and as previously observed (24), there
was no increase in incorporation of radioactivity into Cx43
during a 2-h metabolic labeling interval in cells treated with
dibutyryl cAMP for 6 or 24 h. Thus, angiotensin II but not
cAMP increases the rate of Cx43 protein synthesis in cultured
neonatal rat ventricular myocytes.
Discussion
Regulation of connexin expression by mediators of the
cardiac hypertrophic response. The results of this study indicate that exposure of neonatal ventricular myocytes to angiotensin II for 24 h enhances expression of the principal ventricular gap junction protein, Cx43, and leads to an increase in the
number and size of gap junctions. Because angiotensin has
been implicated as an important mediator of ventricular
remodeling after myocardial infarction, our results suggest that
one potential feature of the remodeling process involves
dynamic changes in gap junction protein expression and distribution, giving rise to anatomic substrates of ventricular
805
arrhythmias. In support of this concept are results of recent
clinical trials showing a significant reduction in the incidence of
sudden death or fatal myocardial infarcts in postinfarct patients treated with angiotensin-converting enzyme inhibitors
(15,30,31).
In previous studies (24), we showed that exposure of
cultured neonatal myocytes to cAMP increases Cx43 content
and the number and size of gap junctions. The results of the
present study, combined with these previous observations (24),
suggest that activation of at least two major signal transduction
pathways, those involving increased intracellular cAMP levels
and those stimulated by angiotensin II, enhance expression of
Cx43 and increase the number and size of gap junctions. The
dynamic nature of these changes suggests that remodeling of
conduction pathways during early phases of infarct healing is
an active process involving enhanced connexin expression and
rearrangements of gap junction distributions rather than being
a purely passive process in which some junctions become
disrupted by the accumulation of interstitial fibrosis and others
are preserved.
Methodologic considerations and limitations. Our studies
were performed in cultures of neonatal rat ventricular myocytes using angiotensin concentrations similar to those used in
previous studies by others (18,20,21,23). It is possible that the
effects of angiotensin II could be different in neonatal cells that
are actively synthesizing muscle-specific genes than in fully
developed adult myocytes. However, there is evidence to
suggest that angiotensin II induces a hypertrophic response in
adult myocardium. For example, chronic infusion of subpressor amounts of angiotensin II causes ventricular hypertrophy in
rats without changing blood pressure (32), and cardiac hypertrophy can be prevented by angiotensin-converting enzyme
inhibitors in rats with abdominal aortic constriction (33). These
observations, combined with clinical results of converting
enzyme inhibitor therapy in patients with heart disease, suggest
that angiotensin II acts as an endogenous growth factor in
heart muscle and that it elicits a hypertrophic response, albeit
by one or more direct and/or indirect mechanisms.
Angiotensin II has been shown to stimulate a hyperplastic
response in cardiac fibroblasts that may also express Cx43
(20,34). It is possible, therefore, that some of the effects
observed in cultures exposed to angiotensin II could have been
due to changes in Cx43 expression in fibroblasts. However,
contamination by fibroblasts in our cultures was modest
(,10%) and fibroblast growth was inhibited by including 0.1
mmol/liter bromodeoxyuridine in the culture medium. Furthermore, the results of confocal immunofluorescence microscopy and electron microscopic morphometry demonstrated
directly that cardiac myocyte gap junctions increased in size
and number in response to angiotensin II. Although a change
in fibroblast Cx43 content probably did not contribute significantly to the increase in total Cx43 signal measured in immunoblots, recent evidence has suggested that the hypertrophic
growth response induced by angiotensin in cardiac myocytes
may be mediated, at least in part, by a paracrine mechanism
involving release of endothelin-1 by cardiac fibroblasts (35).
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DODGE ET AL.
EFFECTS OF ANGIOTENSIN ON Cx43 EXPRESSION
We are not aware of reports on the effects of endothelin-1 on
connexin expression by cardiac myocytes. Future studies will be
required to sort out the complex interactions between cardiac
myocytes and fibroblasts in the development of anatomic
substrates for arrhythmias in heart disease.
Signal transduction pathways regulating connexin expression. The specific signal transduction pathways responsible for
the effects of angiotensin II on Cx43 expression are not known.
Angiotensin has been shown to activate both protein kinase C and
mitogen-activated protein kinase pathways in neonatal cardiac
myocytes (23), either of which could have played a role in
increasing Cx43 expression. It is likely, however, that the responsible pathways activated by angiotensin II are distinct from those
activated by cAMP, suggesting that multiple pathways may affect
connexin expression. Further evidence that angiotensin and
cAMP cause accumulation of Cx43 by disparate mechanisms
comes from the results of metabolic labeling studies. We observed
a marked effect of angiotensin II on the rate of Cx43 protein
synthesis as demonstrated by a nearly threefold increase in the
amount of [35S]methionine incorporated into Cx43 during a 2-h
pulse interval that coincided with the final 2 h of a 24-h exposure
of myocytes to angiotensin II. Interestingly, although the increase
in total Cx43 content (approximately twofold) was equivalent in
myocytes exposed to either angiotensin II or dibutyryl cAMP (24)
for 24 h, no increase in Cx43 synthesis rate was observed in cells
treated with cAMP. These observations suggest that accumulation of Cx43 in response to prolonged cAMP exposure is related
to diminished Cx43 proteolysis. This potential mechanism is
consistent with results of earlier studies demonstrating that cAMP
delays the disappearance of gap junctions in freshly isolated pairs
of rat hepatocytes (36), in which loss of intercellular coupling
typically occurs with a time course similar to the measured
half-life of the major hepatocyte gap junction protein, Cx32 (37).
Conclusions. The results of this study suggest that connexin expression is activated during a generalized hypertrophic
response and may be regulated by multiple signal transduction
pathways and involve different molecular mechanisms. A more
detailed understanding of these pathways and mechanisms
could provide specific targets for new therapies designed to
limit structural alterations that create anatomic substrates of
lethal ventricular arrhythmias in patients with heart disease.
We thank Susan Johnson for secretarial assistance.
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