1 Center for Research on Reproduction and Women's Health, University of
Pennsylvania School of Medicine, 1355 Biomedical Research Building II/III, 421
Curie Blvd., Philadelphia, PA 19104, USA
2 Department of Pathology, University of Pennsylvania School of Medicine, 1355
Biomedical Research Building II/III, 421 Curie Blvd., Philadelphia, PA 19104,
USA
* These authors contributed equally to this work
Present address: GlaxoSmithKline Pharmaceuticals, Collegeville, PA 19426,
USA
Author for correspondence (e-mail:
radice{at}mail.med.upenn.edu
)
Accepted 21 January 2002
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Summary |
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Key words: Transgenic, Cell adhesion, Cardiac hypertrophy, Cyclin D1, DNA synthesis
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Introduction |
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Classical cadherins are a family of cell surface glycoproteins that mediate
calcium-dependent cell-cell adhesion primarily in a homophilic manner
(Vleminckx and Kemler, 1999).
The classical cadherins are single pass transmembrane proteins comprising five
extracellular domains, a transmembrane domain and a cytoplasmic domain. The
cadherins form cisdimers in the plasma membrane that interact in an
anti-parallel fashion with like cadherins on a neighboring cell, creating an
adhesion zipper between the cells (Shapiro
et al., 1995
). Cadherin adhesive activity is regulated by a group
of proteins belonging to the catenin family that bind to the conserved
cytoplasmic domain of the cadherin
(Gumbiner, 2000
).
ß-Catenin or
-catenin (plakoglobin) bind directly to the
C-terminal region of the cadherin, whereas p120 interacts with the
juxtamembrane region (Anastasiadis and
Reynolds, 2000
).
-Catenin binds to ß- or
-catenin, which links the cadherin/catenin complex either directly
(Rimm et al., 1995
) or
indirectly (Knudsen et al.,
1995
; Watabe-Uchida et al.,
1998
) to the actin cytoskeleton. Recently, a novel catenin,
T-catenin, was found to be expressed at high levels in the heart, where
it localized to the intercalated disc
(Janssens et al., 2001
).
Cadherin family members have distinct spatial and temporal patterns of
expression during embryonic development and in the adult
(Takeichi, 1995). N-cadherin
is widely expressed in the early postimplantation embryo
(Radice et al., 1997
)
including the precardiac mesoderm and continues to be expressed at high levels
in embryonic, fetal and adult myocardium
(Angst et al., 1997
).
N-cadherin is found in other tissues such as skeletal muscle, which expresses
multiple cadherin subtypes including R- and M-cadherin
(Kaufmann et al., 1999
). By
contrast, E-cadherin is not expressed in muscle, but mainly found in epithelia
throughout the body. In adult myocardium, N-cadherin/catenin complex is
primarily localized to adherens junctions in intercalated discs where it
serves as an attachment site for myofibrils. In addition, N-cadherin is found
in extrajunctional sites localized to periodic bands along the lateral
membrane referred to as costameres
(Goncharova et al., 1992
). The
addition of function blocking antibodies to chick myocyte culture has
demonstrated the importance of N-cadherin in cell-cell interaction and
myofibril organization (Goncharova et al.,
1992
; Peralta Soler and
Knudsen, 1994
). Furthermore, injection of cDNA encoding a
truncated N-cadherin molecule lacking its extracellular domain (i.e.
dominant-negative) caused cells to lose contact with their neighbors and
myofibril organization was disrupted
(Hertig et al., 1996
).
N-cadherin has been implicated in several aspects of cardiac development
including sorting out of the precardiac mesoderm
(Linask et al., 1997
),
establishment of left-right asymmetry
(Garcia-Castro et al., 2000
),
cardiac looping morphogenesis (Shiraishi
et al., 1993
), and trabeculation of the myocardial wall
(Ong et al., 1998
). Using gene
targeting technology, we previously demonstrated that loss of N-cadherin
resulted in embryonic lethality associated with multiple developmental
abnormalities including a severe cardiovascular defect
(Radice et al., 1997
).
Recently, we generated chimeric embryos with N-cadherin double-knockout ES
cells demonstrating that N-cadherin-mediated adhesion was essential for
maintaining myocyte interactions during the morphological transition from an
epithelial to compacted myocardial cell layer
(Kostetskii et al., 2001
).
Taken together, these data indicate that N-cadherin plays a critical role in
myocardial development and function.
There is limited information pertaining to the consequences of ectopic or
overexpression of cadherins on tissue homeostasis. Importantly, recent data
indicate that misexpression of cadherins influences cellular behavior of tumor
cells. Ectopic expression of N-cadherin in squamous cell carcinoma cell lines
(Islam et al., 1996) as well
as breast cancer cells (Hazan et al.,
2000
; Nieman et al.,
1999
) either in the presence or absence of endogenous E-cadherin
leads to increased invasiveness in vitro and in vivo. Furthermore,
misexpression of E-cadherin in retinal pigment epithelial (RPE) cell lines
affects the distribution of polarized proteins such as
Na+,K+-ATPase and the expression of cytoskeletal
proteins (Marrs et al., 1995
).
In a different experimental system, overexpression of E-cadherin in the
epithelial intestinal crypts of chimeric mice decreased the cellular migration
up the villus (Hermiston et al.,
1996
). Taken together, these studies indicate that cadherin
function is dependent on cellular context as well as levels of expression.
To address the role of cadherin subtype specificity on cardiac development
and function, we generated transgenic mice expressing either N- or E-cadherin
in the heart (Luo et al.,
2001). Ectopic expression of E-cadherin in the myocardium did not
interfere with normal cardiac development consistent with its ability to
restore myocyte adhesion and cardiac morphogenesis in N-cadherin-null embryos
(Luo et al., 2001
). However,
both overexpression of N-cadherin or misexpression of E-cadherin in the adult
myocardium caused dilated cardiomyopathy, which was probably caused by
perturbation of normal intercalated disc function. Ectopic expression of the
epithelial cadherin resulted in earlier onset of the phenotype and increased
mortality compared with mice that overexpress N-cadherin. Furthermore,
misexpression of E-cadherin induced DNA synthesis in the absence of
cytokinesis in myocytes normally withdrawn from the cell cycle; this resulted
in an increased number of `binucleated' myocytes in the transgenic heart. Our
results indicate that modulation of cadherin-mediated adhesion leads to
dilated cardiomyopathy in mice with pathological features similar to those
found in human patients with end stage heart failure.
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Materials and Methods |
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Immunohistochemistry and histological analysis
Hearts were fixed overnight in freshly prepared formalin in PBS, pH 7.4.
After rinsing in PBS, the hearts were dehydrated and embedded in paraffin wax
according to standard procedures. Sections (6 µm) were cut, mounted,
dewaxed in xylene, rehydrated through an ethanol series, and then heated in
1x Antigen Unmasking Solution (Vector Laboratories, Burlingame, CA) in a
microwave oven (350 watts) for 10 minutes to unmask the epitope. The sections
were washed in PBS before blocking with 5% nonfat milk/PBS for 30 minutes.
Antibodies were diluted in 5% nonfat milk/PBS as follows: mouse monoclonal
anti N-cadherin, 1:100 (3B9; Zymed, So. San Francisco, CA); mouse monoclonal
anti E-cadherin, 1:100 (4A2C7; Zymed, So. San Francisco, CA); mouse monoclonal
anti-ß-catenin, 1:100 (CAT-5H10; Zymed); rabbit polyclonal anti-connexin
43, 1:100 (Zymed); mouse monoclonal anti-cyclin D1, 1:50 (sc-450; Santa Cruz
Biotechnology, Santa Cruz, CA); mouse monoclonal anti-vinculin, 1:500 (a kind
gift from Joe Sanger, University of Pennsylvania), rat monoclonal anti-chicken
N-cadherin, 1:100 (NCD-2, a kind gift from Masatoshi Takeichi, Kyoto
University, Kyoto, Japan). Samples were incubated overnight at 4°C with
primary antibodies. After washing in PBS, sections were incubated with
biotinylated anti-mouse or anti-rat IgG or FITC-conjugated goat anti-mouse,
1:500 (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 hour at room
temperature. After washing in PBS, if necessary, sections were incubated with
Cy3-conjugated strepavidin for 30 minutes at room temperature. The sections
were washed in PBS, and mounted for analysis with a confocal microscope. For
nuclear staining, the slide mounting solution contained DAPI (4',
6-diamidino-2-phenylindole, 1:500, Sigma, St Louis, MO). Quantification of
binucleated cells was performed with Openlab software (Improvision).
For histological analysis, hearts were isolated at different ages, fixed in
formalin, processed for paraffin sectioning, and stained with either
Hematoxylin and Eosin or Alizarin Red. Ultrastructural analysis of myocardium
was performed by transmission electron microscopy as previously described
(Lavker et al., 1991).
Western immunoblotting
Freshly isolated hearts were washed in cold PBS and then homogenized in
extraction buffer (150 mM NaCl, 50 mM Tris-Cl, pH 7.2, 1% Triton, 0.1% SDS,
0.5% deoxycholic acid, sodium salt), Homogenates were subjected to SDS-PAGE,
and the resolved proteins were transferred to nitrocellulose by a semi-dry
transfer system. After blocking with 5% nonfat milk for 2 hours, the blots
were washed in TBS-T pH 7.6, and incubated for 1 hour at room temperature with
primary antibodies. Antibodies were diluted in 5% nonfat milk/PBS as follows:
mouse monoclonal anti-N-cadherin, 1:1000; mouse monoclonal anti E-cadherin,
1:1000; mouse monoclonal anti-ß-catenin, 1:1000; rabbit polyclonal
anti-connexin 43, 1:1000; mouse monoclonal anti-vinculin, 1:5000; rat
monoclonal anti-chicken N-cadherin, 1:500; mouse monoclonal anti-rabbit GAPDH,
1:4000 (RDI, Flanders, NJ). The blots were washed in TBS-T and incubated with
secondary antibodies: alkaline phosphatase-conjugated anti-mouse, anti-rat or
anti-rabbit IgG (1:1000, Jackson ImmunoResearch Laboratories, West Grove, PA),
for 1 hour at room temperature. After washing with TBS-T, the bound antibodies
were detected using enhanced chemifluorescence (Vistra ECF kit; Amersham,
Arlington Heights, IL). Chick embryonic heart and human keratinocyte lysates
were used as positive controls for anti-chick N-cadherin and anti-human
E-cadherin antibodies, respectively. Variations in sample loading were
normalized relative to GAPDH signal. Labeled blots were analyzed using Storm
860 Imaging system and Imagequant software (Molecular Dynamics, Sunnyvale,
CA).
Northern blot analysis
Total heart RNA was collected from transgenic and nontransgenic littermates
and purified with Tri Reagent (Sigma). RNA (15 µg) was separated on a 1.5%
formadehyde agarose gel, transferred to Zeta-Probe (Bio-Rad) blotting membrane
in 20x SSC solution overnight. After transferring, the blot was
crosslinked and prehybridized with QuickHyb solution (Stratagene) at 68°C
for 20 minutes. The rat ANF cDNA probe (a kind gift from Jason Rogers,
Washington University, St Louis, MO) and GAPDH cDNA probe (Clontech) were
labeled with 32P using DNA labeling beads (Amersham) and purified.
The blot was hybridized with the labeled probes, washed, and analyzed using
the Storm 860 Imaging system and Imagequant software.
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Results |
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The generation of the MHC/Ncad and
MHC/Ecad transgenic lines
was described previously (Luo et al.,
2001
). Comparison of protein expression in the heart of different
transgenic mice indicated high expression in several lines
(Fig. 1A). An additional
smaller band (approximately 105 kDa) observed in the higher expressing
MHC/Ecad lines has been observed previously
(Yoshida-Noro et al., 1984
)
and may correspond to a degradation product. To confirm that expression of the
transgene was restricted to the heart, western blot analysis was performed on
the following noncardiac tissues: brain, kidney, liver, lung and spleen, from
the
MHC/Ncad4 and
MHC/Ecad10 lines. No expression was observed
in noncardiac tissues (data not shown) consistent with previous reports with
the
MHC promoter (Gulick et al.,
1991
). Mice hemizygous for either the
MHC/Ncad or
MHC/Ecad transgenes were mated with wild-type animals. A number of pups
from
MHC/Ecad matings died between one and two weeks after birth. In
comparison, no significant neonatal lethality was observed in the
MHC/Ncad lines. The loss of
MHC/Ecad pups was further
substantiated by examining the genotypes of the animals that survived to
weaning age. Mendelian inheritance predicts that 50% of the mice will inherit
the transgene, which was the case for the
MHC/Ncad transgene (line N4,
125 transgenic:126 nontransgenic). By contrast, mice expressing comparable
levels of E-cadherin were under-represented at weaning (line E33, 75
transgenic:126 nontransgenic, P<0.02). Furthermore, several
MHC/Ecad mice died suddenly during handling.
|
Both transgenic animals exhibited aberrant cardiac morphology; however, the
phenotype was more pronounced in the surviving MHC/Ecad mice, in
comparison to the
MHC/Ncad mice, as demonstrated by increased dilation
of the atria and ventricles (Fig.
1B). Comparison of the heart weight to body weight ratios of
MHC/Ncad and surviving
MHC/Ecad mice illustrates this point
further (line N4 versus line E33, Fig.
1C). Mice (<100 days old) expressing high levels of exogenous
cadherin in the heart displayed a significant increase in heart weight:body
weight ratio compared with wild-type mice consistent with a hypertrophic
response (Fig. 1C). This
phenotype was associated with increased postnatal lethality especially among
the
MHC/Ecad mice. Females positive for either the
MHC/Ecad or
MHC/Ncad transgene had difficulty during pregnancy and nursing;
therefore, the transgene was transmitted through the transgenic males. Cardiac
hypertrophy was not observed in the lower expressing
MHC/Ncad and
MHC/Ecad mice indicating an association between transgene expression
levels and the cardiac phenotype. Taken together, our data are consistent with
a more severe dilated phenotype observed in the surviving
MHC/Ecad mice
in comparison with the
MHC/Ncad.
E-cadherin localizes to the intercalated disc in cardiomyocytes
To determine the cellular localization of cadherins in the myocardium,
immunohistochemistry was performed on hearts from 4-week-old transgenic mice.
Consistent with previous reports (Angst et
al., 1997), endogenous N-cadherin localizes predominantly to the
intercalated disc in wild-type hearts with little cytoplasmic staining
(Fig. 2A). N-cadherin
immunostaining in the
MHC/Ncad transgenic heart represents either
chicken N-cadherin alone (Fig.
2B) or mouse and chicken N-cadherin together
(Fig. 2C). As predicted, the
cadherin staining patterns were identical with both antibodies
(Fig. 2B,C), as shown by the
merged images (Fig. 2D).
Similar to endogenous N-cadherin, the chicken protein also localized to the
intercalated disc (Fig. 2B). In
addition, excess exogenous N-cadherin was found distributed throughout the
cytoplasm (Fig. 2B). The
wild-type heart was negative for E-cadherin expression
(Fig. 2E), as was the case for
endogenous E-cadherin (data not shown). By contrast, human E-cadherin, like
endogenous N-cadherin (Fig.
2G), localized to the intercalated disc in the
MHC/Ecad
transgenic heart (Fig. 2F). The
merged images demonstrate that both exogenous E-cadherin and endogenous
N-cadherin were colocalized to the intercalated disc
(Fig. 2H). Both N- and
E-cadherin were observed in the cytoplasm of these transgenic hearts
(
Ncad4 and
Ecad33), whereas lower expressing transgenic lines
showed only intercalated disc staining (data not shown) similar to the
endogenous N-cadherin pattern. This suggests that the intercalated disc was
saturated with cadherin protein in the high expressing lines leading to its
accumulation in the cytoplasm. These data show that the epithelial cadherin,
E-cadherin, is correctly localized to the intercalated disc structure in heart
muscle along with endogenous N-cadherin.
|
E-cadherin stimulates DNA replication in terminally differentiated
myocytes
Histologic analysis of hearts from 4-week-old mice demonstrated the
severity of the phenotype observed in the transgenic hearts
(Fig. 3A-C). An enlarged left
ventricle was observed in the MHC/Ncad heart, whereas both ventricles
were severely dilated in the
MHC/Ecad heart. In addition, dilated
thin-walled atria were observed in the pumpkin-shaped
MHC/Ecad heart
(Fig. 3C). Both the
MHC/Ncad and
MHC/Ecad transgenic hearts contained enlarged
hyperchromatic myocyte nuclei with increased myofibrillar width consistent
with cardiac hypertrophy (Fig.
3D-F). At two months of age, left atrial thrombosis was observed
in the transgenic mice (Fig.
3G). Furthermore, white spots or streaks were often observed in
the atria and ventricles of the transgenic hearts (not shown). Alizarin Red
staining indicated that these white spots correspond to regions of
calcification in the heart (Fig.
3H). Limited regions of fibrosis were observed in older transgenic
animals (data not shown). The thrombosis and calcification were observed in
both
MHC/Ncad (not shown) and
MHC/Ecad transgenic mice; however,
misexpression of E-cadherin often caused earlier onset and a more severe
phenotype compared with overexpression of N-cadherin.
|
The molecular mechanism(s) that control cell cycle withdrawal in terminally
differentiated myocytes is poorly understood. In the case of MHC/Ecad
hearts, we observed many hypertrophic myocytes with two enlarged nuclei side
by side (Fig. 3F), which
suggested that DNA replication and nuclear division had occurred but
cytokinesis had not taken place. The consistent proximal positioning of the
two nuclei suggested that this phenomena did not result from fusion of two
myocytes, but that the nuclei originated from the same cell. The phenotype,
referred to here as `binucleated', was easily visualized by DAPI staining the
heart sections for nuclear DNA (Fig.
4). Karyokinesis often was not complete, resulting in myocytes
with two nuclei not completely separated
(Fig. 4C). By contrast, no
significant increase in `binucleated' myocytes was observed in transgenic
hearts expressing N-cadherin (4% versus 27%) indicating that the phenotype was
caused specifically by E-cadherin (Fig.
4D). The `binucleated' phenotype was observed in both atrial and
ventricular myocytes beginning postnatally at 1 week of age coincident with
increased E-cadherin expression (data not shown). This phenotype was observed
in the three highest expressing
MHC/Ecad transgenic lines (E10, E22,
E33). In comparison, the number of `binucleated' myocytes did not increase
significantly in the lower expressing lines (E9, E12) indicating that the
E-cadherin effect was dosage dependent. The cell-cycle-dependent kinase,
cyclin D1, plays an important role in the initiation of DNA synthesis,
therefore we examined its expression in the transgenic hearts. Consistent with
the `binucleated' phenotype, an increase in cyclin D1 expression was observed
in the
MHC/Ecad heart (Fig.
4F) in comparison to
MHC/Ncad
(Fig. 4E) and wild-type (data
not shown) hearts. To determine whether E-cadherin affected myocyte
proliferation in the embryonic myocardium, we examined the cell proliferation
rate at E10.5 using BrdU labeling in utero. No significant overall difference
in BrdU incorporation was observed between transgenic and wild-type animals
(data not shown). The effect of E-cadherin on myocyte proliferation appears to
be restricted to postnatal myocytes, which normally withdraw from the cell
cycle.
|
Downregulation of connexin 43 and redistribution of vinculin in
MHC/Ecad transgenic hearts
The intercalated disc consists of different junctions, including the
adherens junction, desmosome and gap junction, which function cooperatively to
maintain normal cell-cell interactions between working myocytes. We examined
the expression and cellular localization of several components of these
junctions in hearts of 4-week-old transgenic mice. ß-Catenin localizes
predominantly to the intercalated disc in wild-type hearts with very little
cytoplasmic staining (Fig. 5A).
In comparison, increased staining for ß-catenin in the intercalated disc
and cytoplasm was observed in both MHC/Ncad
(Fig. 5B) and
MHC/Ecad
(Fig. 5C) transgenic hearts.
The increase in ß-catenin was consistent with the increase in total
cadherin observed in the transgenic hearts
(Fig. 2). This coordinated
regulation of cadherin and catenin has been observed by others in vitro
(Nagafuchi et al., 1991
;
Redfield et al., 1997
) and in
vivo (Hermiston et al., 1996
).
A small increase in
-catenin and no significant increase in
-catenin (plakoglobin), which are additional components of the
cadherin/catenin adhesion complex, were observed in the transgenic hearts
(data not shown). In addition, the desmosome marker, desmoplakin, was not
changed significantly in the transgenic hearts (data not shown). The gap
junction protein, connexin 43 (Cx43), is normally localized to the
intercalated disc in the mature myocardium, as shown in wild-type hearts
(Fig. 5D). In the transgenic
hearts, the punctate Cx43 staining was reduced in both
MHC/Ncad
(Fig. 5E) and to an even
greater extent in
MHC/Ecad (Fig.
5F) hearts. Vinculin was associated with the intercalated disc and
Z bands in the wild-type heart (Fig.
5G). A similar pattern was observed in the
MHC/Ncad heart
except that the intercalated disc staining was more intense
(Fig. 5H) consistent with more
functional cadherin/catenin adhesion complexes. However, in the
MHC/Ecad heart, less vinculin was found in the intercalated disc;
instead, it was redistributed to the cytoplasm and showed a striated and
diffuse pattern (Fig. 5I). Both
downregulation of Cx43 and increased vinculin in the cytoplasm are associated
with cardiac hypertrophy and heart failure in humans
(Dupont et al., 2001
;
Peters et al., 1993
;
Schaper et al., 1991
) and
animal models (Wang and Gerdes,
1999
).
|
Changes in protein expression associated with cardiac hypertrophy in
the cadherin transgenic mice
To determine how changes in protein expression correlated with the
cadherin-induced hypertrophy response, western blot analysis was performed on
hearts from 3-day- to 4-week-old wild-type and transgenic mice
(Fig. 6). The time course of
expression of the MHC/Ncad and
MHC/Ecad transgenes was
consistent with previous reports using this cardiac promoter increasing
postnatally in the ventricular myocardium
(Palermo et al., 1996
;
Subramaniam et al., 1991
). As
expected, the mouse and chicken N-cadherin proteins migrate together;
therefore quantitation relative to endogenous protein was performed by
comparing cadherin levels in wild-type and transgenic littermates. Since
N-cadherin and E-cadherin were distinguishable on the blot, the relative
levels of endogenous and exogenous cadherin were compared directly.
Quantitation of the Western blots indicated that both transgenes were
expressed at approximately the same levels (2.3,
Ncad4 versus 2.6,
Ecad10) relative to endogenous N-cadherin, demonstrating that
quantitative differences in exogenous cadherin were most probably not
responsible for the more severe cardiac phenotype observed in the
MHC/Ecad mice. The expression of E-cadherin had no significant effect
on endogenous N-cadherin levels. In both
MHC/Ncad and
MHC/Ecad
hearts, ß-catenin levels increase concomitantly with exogenous cadherin.
By contrast, ß-catenin levels normally decrease with age in wild-type
littermates. Since plakoglobin is capable of binding to the cytoplasmic domain
of N-cadherin, we examined plakoglobin levels in the transgenic hearts;
however, no significant change in expression was observed (data not shown).
The gap junction protein, Cx43, showed a remarkable reduction in
MHC/Ecad mice in comparison to
MHC/Ncad mice at 4 weeks of age,
consistent with the more severe hypertrophic response. The degree of
downregulation of Cx43 correlated with the severity of the hypertrophy and
varied among animals from the same transgenic line. By contrast, vinculin
levels appeared similar in wild-type and transgenic hearts indicating that the
increased cytoplasmic staining in the
MHC/Ecad hearts probably
represented a redistribution away from the cell surface, which has been
observed in human cardiac hypertrophy
(Schaper et al., 1991
). In
addition, F-actin protein levels did not change in the transgenic mice (data
not shown) suggesting that increased cadherin expression did not alter the
actin cytoskeleton significantly. Taken together, the more severe
perturbations of intercalated disc proteins in the
MHC/Ecad mice were
consistent with an earlier onset and increased mortality observed in
MHC/Ecad animals compared with
MHC/Ncad mice.
|
Modulation of cadherin expression in the heart leads to upregulation
of ANF
To determine the time of onset and severity of the cardiac phenotype in
MHC/Ncad and
MHC/Ecad mice, northern blot analysis was performed
on heart samples to examine expression of the hypertrophy marker, ANF
(Vikstrom et al., 1998
), in
3-day- to 4-week-old wild-type and transgenic mice
(Fig. 7A). Both
MHC/Ncad
and
MHC/Ecad transgenic mice displayed increased ANF expression
compared with wild-type. However, ANF increased earlier and to a greater
extent in
MHC/Ecad mice consistent with the severity of the
hypertrophic response in these animals. The increase in ANF expression was
already significant by one week after birth and was consistent with the early
postnatal lethality observed in the
MHC/Ecad line
(Fig. 7B).
|
Electron microscopic analysis shows well-organized myofibrillar
structures in the E-cadherin transgenic hearts
To examine myofibril organization and cell-cell contacts more closely,
transmission electron microscopy was performed on hearts from wild-type and
transgenic 4-week-old mice. Ultrastructural analysis showed that wild-type,
MHC/Ecad, and
MHC/Ncad (data not shown) hearts had
well-organized myofibrils with clearly defined Z bands
(Fig. 8). The intercalated disc
structures appeared well formed, and insertion of the myofibrils into the
adherens junction appeared normal. In summary, the misexpression of cadherins
did not result in any gross morphological abnormalities in the myocardium of
the transgenic animals.
|
![]() |
Discussion |
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The cadherin superfamily can be divided into several subfamilies including
the classical cadherins, which consist of approximately 20 members in mammals.
Structural and biochemical studies suggest that cadherins form cis-dimers that
interact in an anti-parallel fashion with a like cadherin pair on an adjacent
cell. This configuration is repeated forming a zipper-like structure thus
establishing an adherens junction between the cells. It is thought that
tissue-specific expression of different cadherin subtypes is critical for
normal tissue development and function. Most cell types including skeletal
muscle express multiple cadherin subtypes; however, cardiac muscle depends
primarily on one classical cadherin, N-cadherin. By contrast, E-cadherin is
normally not expressed in muscle, but found in most epithelia throughout the
body. The chick and mouse N-cadherin proteins show high overall amino acid
similarity (92%) including the nearly identical (99%) transmembrane and
cytoplasmic domains. The ability of these N-cadherin proteins to interact in
trans was demonstrated in a cell aggregation assay using L-cells transfected
with cDNA encoding either chick or mouse N-cadherin
(Miyatani et al., 1989). In
addition, it is likely that these cadherin homologs generate cis-dimers, which
produce an interspecies chimeric zipper structure. The cis interaction is
likely given the extent of amino acid similarity (92%) and the fact that N-
and R-cadherin, which are less well conserved (74% similarity), interact to
form lateral dimers (Shan et al.,
2000
). By contrast, mouse N- and E-cadherin show 49% amino acid
similarity overall and in vitro studies indicate that N- and E-cadherin do not
interact in either cis or trans (Miyatani
et al., 1989
; Shan et al.,
2000
).
In adult myocardium, N-cadherin/catenin complex is primarily localized to
adherens junctions in intercalated discs where it serves as an attachment site
for myofibrils, in addition to its structural role in maintaining myocyte
adhesion. Increased cadherin expression in transgenic hearts was accompanied
by increased steady-state levels of ß-catenin, also depicted by increased
immunolocalization of cadherin/catenin complexes to the intercalated disc. How
might cadherin misexpression effect myocyte interactions? Several possible
factors affecting intercalated disc function are considered. We speculate that
excess cadherin/catenin complexes compared with myofibrils may alter the
contractile dynamics by changing the stoichiometry of the cadherin/myofibril
connection leading to less efficient force transduction across the plasma
membrane (Fig. 9). The working
heart is under tremendous mechanical load, therefore it is difficult to rule
out the possibility that subtle differences between mouse and chick N-cadherin
may structurally perturb the adhesion zipper. In comparison, the adherens
junction in MHC/Ecad mice consists of two different cadherin/catenin
complexes that may further perturb the contractile dynamics since E-cadherin
cannot interact with N-cadherin and differences in the cytoplasmic domain may
alter myofibril connections. In both cases, the dissipation of the contractile
force across the plasma membrane leads to a compensatory response (i.e.
hypertrophy) with the greater effect caused by introduction of the epithelial
cadherin. Since both transgenes are expressed at approximately similar levels,
a qualitative verses quantitative difference in cadherin-mediated adhesion is
probably responsible for the severe cardiac hypertrophy observed in the
E-cadherin transgenic mice.
|
The increased penetrance, earlier onset, and increased severity of the
cardiac phenotype in MHC/Ecad mice suggests that the epithelial
cadherin may further perturb the transmission of the contractile force across
the intercalated disc structure. In contrast to chick N-cadherin, the less
similar E-cadherin is more likely to interfere with adherens junction
organization by intercalating into the cadherin zipper, thus disrupting the
normal homogeneous clustering of N-cadherin/catenin complexes
(Fig. 9). Mouse and chicken
N-cadherin are nearly identical in the cytoplasmic and transmembrane domains;
therefore, we predict normal interaction(s) with the submembranous myofibril
connection. In comparison, the cytoplasmic domain of E-cadherin is less
similar (61%) and somewhat shorter (10 amino acids) than N-cadherin. Although
TEM did not indicate any obvious defect in myofibril insertion at the cell
membrane, it is possible that subtle molecular differences in the
E-cadherin/myofibril connection may lead to contractile dysfunction in the
MHC/Ecad heart. In summary, E-cadherin may be acting as a
dominant-negative cadherin in heart muscle by altering the cadherin zipper
structure leading to less efficient contractile force transduction across the
plasma membrane.
Terminally differentiated cardiac myocytes normally withdraw from the cell
cycle after birth. Therefore, we were surprised that E-cadherin misexpression
in the postnatal mouse heart led to `binucleated' myocytes; this phenotype was
not observed in the N-cadherin transgenic animals. In addition to its role in
cell adhesion, ß-catenin in concert with the TCF/LEF family of
transcription factors is capable of regulating cell cycle progression by
transcriptionally activating genes such as cyclin D1
(Tetsu and McCormick, 1999).
However, increased expression of ß-catenin is probably not responsible
for cyclin D1 expression in our in vivo model since ß-catenin is
upregulated in both N- and E-cadherin transgenic mice, and the `binucleated'
phenotype is observed only in the E-cadherin animals. Although E-cadherin was
expressed throughout the myocardium, a relatively small fraction of the
myocytes exhibited the `binucleated' phenotype. This suggests that expression
of other factors required for DNA replication may be limited to a subset of
cardiomyocytes present in the postnatal heart. A multinucleation phenotype
without hypertrophy was observed previously in
MHC/cyclin D1 transgenic
mice (Soonpaa et al., 1997
)
consistent with the role of cyclin D1 in cell cycle progression; however, it
is unclear how E-cadherin induces cyclin D1 expression in myocytes. In
contrast to our findings, epithelial cell proliferation decreased when
E-cadherin was overexpressed in the intestinal crypts of transgenic mice
(Hermiston et al., 1996
) and
in mammary carcinoma cells (St Croix et
al., 1998
) demonstrating that cadherin `signaling' activity is
dependent on cellular context. Interestingly, in the adult, normally
N-cadherin protein levels are highest in the heart and brain, two tissues that
express little or no E-cadherin. Normally, N-cadherin-expressing myocytes and
neurons withdraw from the cell cycle following terminal differentiation,
whereas E-cadherin-expressing epithelial cells maintain their capacity to
undergo cell division in the adult. The ability of E-cadherin to stimulate DNA
synthesis in myocytes, in contrast to N-cadherin, suggests that N-cadherin may
normally provide growth inhibitory signals to regulate cell cycle progression
in the postnatal heart. It is intriguing to speculate that different cadherin
subtypes may regulate cell cycle progression in differentiated tissues. A
69-amino acid region of EC4 of N-cadherin was recently shown to be sufficient
to promote both an epithelial to mesenchyme transition in squamous epithelial
cells and increased cell motility (Kim et
al., 2000
). In future studies, it will be of interest to determine
whether the cytoplasmic or extracellular domain of E-cadherin is responsible
for the `binucleated' phenotype.
The intercalated disc acts as an organizing center for the adjoining
myocytes with different junctional complexes performing specific inter-related
functions. Structural perturbation of the intercalated disc accompanied by
downregulation of N-cadherin was observed in a hereditary hamster model of
dilated cardiomyopathy (Fujio et al.,
1995). Furthermore, in a chronic pressure overload model,
ß-catenin was redistributed from the plasma membrane to the cytoplasm;
however, there was no change in N-cadherin
(Wang and Gerdes, 1999
). The
downregulation of Cx43 and increased cytoplasmic localization of vinculin in
the E-cadherin transgenic mice are consistent with observations made in
patients with chronic heart failure
(Peters et al., 1993
;
Schaper et al., 1991
). The
dramatic effect on Cx43 expression is probably not caused by cadherin
overexpression per se, but due to the severe hypertrophy response caused by
ectopic expression of E-cadherin in the myocardium.
The ability of cancer cells to alter their cadherin profile suggests that
other diseases may also be affected by inappropriate cadherin expression. In
the future, it will be interesting to examine cardiac tissue from patients to
determine whether pressure overload, viral infection, and other insults may
lead to inappropriate expression of E-cadherin. For example, misexpression may
result from demethylation of the E-cadherin promoter and its subsequent
transcriptional activation in cardiomyocytes. Furthermore, the bacterium
Listeria monocytogenes uses human E-cadherin as a receptor. Recently,
a transgenic mouse model for Listeria infection was generated by
expressing human E-cadherin in the intestinal epithelium
(Lecuit et al., 2001). It will
be interesting to determine whether our animal model may provide a novel
paradigm to examine bacterial infection in the heart.
Cell-cell and cell-extracellular matrix interactions are important for
tissue homeostasis. Altering cadherin-mediated adhesion, as demonstrated here,
had a pronounced effect on cardiac pathology. In contrast, similar transgenic
experiments expressing wild-type 5 integrin, a fibronectin receptor,
did not interfere with heart function. However, in the same study an
5
integrin molecule with its cytoplasmic domain deleted (i.e. gain of function)
caused severe cardiac hypertrophy
(Valencik and McDonald, 2001
).
Furthermore, proteins involved in regulation of the actin cytoskeleton, such
as the small GTP-binding protein rac1 and the muscle LIM protein MLP, have
been implicated in cardiomyopathy in mice
(Arber et al., 1997
;
Sussman et al., 2000
) and
humans (Zolk et al., 2000
).
Taken together, the data suggest that dysregulation of either cadherin or the
actin cytoskeleton can lead to impaired force transmission and dilated
cardiomyopathy.
In summary, we provide the first evidence that altering the cadherin composition of the intercalated disc can lead to cardiomyopathy and that E-cadherin, but not N-cadherin, can stimulate DNA synthesis in cardiomyocytes normally withdrawn from the cell cycle.
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