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J Biol Chem, Vol. 274, Issue 52, 37362-37369, December 24, 1999
From the Cardiac chamber morphogenesis requires the
coordinated growth of both cardiac muscle and endocardial cell
lineages. Paracrine growth factors may modulate the coordinated
cellular specification and differentiation during cardiac chamber
morphogenesis, as suggested by the essential role of
endothelial-derived growth factors, neuregulin-1, and insulin-like
growth factor-I. Using the whole mouse embryo culture system for
delivery of diffusible factors into the cardiac chamber, neuregulin-1
was shown to promote trabeculation of the ventricular wall. Another
factor, insulin-like growth factor-I, had no apparent effect by itself.
Combined treatment with neuregulin-1 and insulin-like growth factor-I
strongly induced DNA synthesis of cardiomyocytes and expansion of both
the ventricular compact zone and the atrioventricular cushions leading
to chamber growth and maturation. In cultured cardiomyocytes,
combined neuregulin-1 and insulin-like growth factor-I also had a
synergistic effect to promote DNA synthesis and cellular growth, which
were prevented by wortmannin, an inhibitor of phosphatidylinositol
3-kinase. Adenoviral delivery of dominant negative Rac1, which acts
downstream of phosphatidylinositol 3-kinase, blocked the effect of
combined neuregulin-1/insulin-like growth factor-I treatment. These
studies support the concept that the interaction of neuregulin-1 and
insulin-like growth factor-I pathways plays an important role in
coordinating cardiac chamber morphogenesis and may occur through
convergent activation of phosphatidylinositol 3-kinase.
The formation, maturation, and septation of distinct cardiac
chambers during heart development may require the interplay of signals
between myocardial and endocardial cell lineages (1, 2). These distinct
cell types present in the cardiogenic mesoderm will give rise to the
double-layered heart tube, with myocardial cells forming the outer
layer and endocardial cells forming the inner layer (3, 4). Within the
looped cardiac tube, positional signals in the ventricular segment
induce specific maturational steps in ventricular muscle cells,
including trabeculation, expansion of the ventricular compact zone and
atrioventricular cushions, and development of the interventricular
septum. Another set of cues leads to the generation of cushion
mesenchyme derived from endocardial cells, which contributes to
septation of the conotruncus and outflow tract and remodeling of the
atrioventricular canal, which ultimately separates the atrial and
ventricular chambers (1, 2, 5). It has been previously shown by
in vitro assay systems that cushion formation depends on
extracellular matrix, and signals emanating from the adjacent
cardiomyocytes (5, 6). However, the potential effect of
endocardial-derived signals in cushion morphogenesis has not yet been
explored. The maturational steps in ventricular muscle and endocardial
cells might be mutually coordinated. However, the interplaying signals
involved at these different steps of the developing embryonic heart are
still unknown.
Recent studies from targeted gene inactivation in the mouse indicate
that two likely candidates for exerting paracrine effects on
ventricular chamber and cushion development are neuregulin-1 (NRG-1)1 and insulin-like
growth factor-I (IGF-I) (7, 8). Both NRG-1 and IGF-I are
endothelial-derived signals expressed early in embryonic development
that bind and activate receptors expressed throughout the cardiac
chamber (7, 9, 10). Targeted ablation of these growth factors or their
cognate receptors severely affects murine cardiac morphogenesis.
Homozygous null embryos for NRG-1 or the cognate receptors erbB2 or
erbB4 display a lack of trabeculation of the ventricular wall, whereas
individual knockouts for IGF-I or IGF-I receptor die perinatally with
deficient myofibrillogenesis of ventricular myocytes (7, 8,
11-16).
To assess the role of NRG-1 and IGF-I in cardiac chamber maturation and
morphogenesis in intact embryos, we have utilized the whole mouse
embryo culture system for injection of these peptide growth factors
through the ventricular wall of mouse embryos. The distinct advantage
of the ex vivo mouse embryo culture system is that it allows
functional studies of exogenous growth factors during cardiac
morphogenesis in the context of the intact embryo. The present study
documents that NRG-1 induces trabeculation of the ventricular wall
without a significant increase in the proliferation of cardiomyocytes,
in agreement with an essential role of NRG-1 to initiate trabeculae
(7). Another secreted growth factor, IGF-I, which is essential for
cardiac growth in neonatal heart, had no apparent effect on
development. However, combined injection of NRG-1 and IGF-I induced DNA
synthesis in ventricular myocytes and significant growth of the
ventricular compact zone. The potentiation of NRG-1 and IGF-I on
cardiomyocyte proliferation was corroborated in cultured cardiac muscle
cells. To dissect downstream signals activated by NRG-1 and IGF-I, we
used specific inhibitors or dominant negative mutants to block either
the MAP kinase or phosphatidylinositol (PI) 3-kinase signaling pathways
in cultured cardiomyocytes. Our results indicate that the synergistic
effect was mediated by convergent activation of PI 3-kinase rather than
the MAP kinase pathway, which was activated by each growth factor
alone. Taken together, these results support a synergistic role for
NRG-1 and IGF-I in coordinating ventricular chamber growth and
maturation, which may occur through convergent signaling pathways at
the level of PI 3-kinase activation.
Mouse Embryo Culture--
Preparation of rat serum was performed
as described by Cockroft (17). Whole mouse embryos were cultured
according to the method of Sturm and Tam (18) as described previously
(19). Briefly, timed pregnant female C57Bl6 mice maintained and bred in
colonies were sacrificed by cervical dislocation. The uterus was
dissected and rinsed in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, 1.8 mM KH2PO4) to
remove residual blood and then transferred to PB1 medium (137 mM NaCl, 2.7 mM KCl, 0.5 mM
MgCl2, 8.04 mM Na2HPO4,
1.47 mM KH2PO4, 0.9 mM
CaCl2, 0.33 mM sodium pyruvate, 1 g/liter
glucose, 0.01 g of phenol red, pH 7.35, 100 ml/ml streptomycin,
100 unit/ml penicillin; all reagents were from Sigma). Embryos of 11.5 days post-coitum (E11.5) were dissected from the uterus, and the
decidual mass and Riechert's membrane were removed. The embryos were
released from the yolk sac and amnion, which remained attached to
ensure continuity of the vessels connecting the embryo to the yolk sac
or the umbilical vessels from the embryo to the placenta. They were
then transferred to pre-equilibrated medium containing 50% rat serum
and constant gassing (95% O2, 5% CO2) in
roller culture bottles and incubated for the following 26 h. To
confirm specific embryonic stages of murine development we have used
the remodeling of the forelimb buds of cultured embryos as described
previously (19). The smooth distal borders of the handplate at E11.5
are indented by E12.5 (20).
Microinjection Protocol--
After 1 h in culture, the
embryos were placed in a Petri dish and microinjected into the left
ventricle using a 6-µm-diameter glass pipette. The micropipettes were
pulled in a multistage pipette puller (Sutter Instruments, Co., Novato,
CA) using 1-mm glass capillaries. The micropipette was attached to a
MX-110-R 4 axis manual micromanipulator (Newport Instruments, Newport,
CA) via electrode holders. All intracardiac injections were performed with a volume of 1 µl, which proceeded at a low flow rate. All capillaries were calibrated by aspirating 3.2 µl into the needle and
injecting one-third of the total volume into three successive embryos.
In control experiments, PBS was used. The concentration of NRG-1
solutions of the BrdUrd Incorporation and Immunostaining in Cultured
Embryos--
Embryos in culture were grown for the last 5 h in
the presence of 30 µg/ml of BrdUrd added to the culture medium.
Embryos were fixed in 4% paraformaldehyde and embedded in paraffin at a thickness of 5 µm. Immunostaining of nuclear BrdUrd was performed on four sections/embryo treatment using antibodies against BrdUrd coupled to biotin followed by incubation with streptavidin coupled to
peroxidase. The slides were then subjected to light microscopy for
visualization of incorporated BrdUrd, which was detected by the
peroxidase color reaction as indicated in the BrdUrd labeling kit
(Zymed Laboratories Inc., CA). The number of BrdUrd
positive and negative cells were determined in at least two images of
200× magnified fields/section in at least four sections/embryo at a location of the ventricular free wall immediately dorsal to the apex of
the chamber. The average number of BrdUrd positive cells was determined
per 100 cells corresponding to each embryo treatment.
In Situ Hybridization Analysis--
Riboprobes for
acetylcholinesterase cDNA were generated using either T7 or T3
polymerase (22) and radiolabeled in the presence of 35S-UTP
(23). In situ hybridization was performed on 5-µm paraffin sections from mouse embryos according to the method described by Lyons
et al. (24). Equal amounts (10,000 cpm/ml) of
35S-labeled riboprobes were added to each section and
washed after overnight hybridization at 60 °C. The slides were
exposed in D19 Kodak emulsion, counterstained with 0.2% toluidine blue
solution, and mounted in Permount.
Microdissection and Scanning Electron Microscopy--
To assess
internal morphological characteristics, embryos were perfusion fixed,
microdissected, and processed for scanning electron microscopy as
described previously (25). Briefly, embryos used for scanning electron
microscopy were removed from the culture bottles and perfusion fixed
with 2% glutaraldehyde, 1% formaldehyde in 0.1 M
cacodylate buffer. Hearts were then processed to obtain the parietal
and septal views of the right ventricle using a previously described
standardized procedure (26). Primary magnification of scanning electron
micrographs was 160× for atrioventricular (AV) cushion distances and
1000× for analysis of ventricular wall morphology. Measurements of AV
cushion distance were acquired from 160× images at a point midway
between the lateral boundaries of the superior and inferior cushion
pads. Measurements of the thickness of the compact zone were acquired
from the 1000× images at a location of the right ventricular free wall
immediately dorsal to the apex of the chamber. At least three
measurements were averaged from each image for both AV cushion and
compact zone distances.
Generation of Recombinant Adenovirus--
Dominant negative
mutants of Ras (N17), Raf (K375R), Rac1 (N17), activated Rac1 (V12),
and GFP (pEGFP, CLONTECH) were subcloned into
shuttle vectors containing either the RSV (N17Ras) or the cytomegalovirus (K375Rraf, N17Rac1, V12Rac1, and GFP)
promoter/enhancer. Recombinant adenovirus were generated as described
previously (27). All recombinant adenovirus were tested for transgene
expression in cardiomyocytes by reverse transcriptase-polymerase chain
reaction, Western blot, or kinase assays as described (27).
Isolation, Culture, and Treatment of Cardiomyocytes--
Primary
cardiac myocytes were isolated from neonatal rat heart ventricular
chambers and prepared by a Percoll gradient technique as described
previously (28). Cardiomyocytes were densely plated in gelatin-coated
10-cm plates or in chamber slides precoated with 1% gelatin and
laminin (Sigma) as described previously (28). After the first day in
culture medium containing 10% serum, cardiomyocytes were washed and
cultured in minimal medium (4:1 Dulbecco's modified Eagle's
medium/medium 199, 100 units/ml penicillin, 100 µg/ml streptomycin,
and 10 mM glutamine). NRG-1 and/or IGF-I were added to the
culture medium at a concentration of 2.5 and 5 nM,
respectively, in agreement with their maximal activities in
dose-response curves. Cardiomyocytes were grown for 24 h in the
presence of growth factors and for additional 6 h in the presence
of 40 µM BrdUrd to measure DNA synthesis or for 72 h
to follow cardiomyocyte morphological changes. Alternatively,
cardiomyocytes were treated with 1 µM wortmannin or
10 µM PD098059 or infected in serum-free medium for
12 h with adenovirus at a multiplicity of 50-100 particles/cell prior to addition of growth factors and then cultured as described above.
Immunostaining--
Cells were rinsed with 1× PBS and fixed in
4% paraformaldehyde for 10 min. Cells were washed in PBS and
permeabilized with PBS containing 1% bovine serum albumin, 0.2%
Triton X-100 for 15 min. Incubation with the primary antibody myomesin
(a kind gift from Dr. J.-C. Perriard, Institute of Cell Biology,
ETH-Zurich, Switzerland), was performed for 1 h in PBS containing
1% bovine serum albumin. Myocytes were washed in PBS/1% bovine serum
albumin then incubated with secondary antibodies coupled to rhodamine and the fluorescent dye bisbenzimide (Hoechst dye 22358) 1 µg/ml for
30 min. The slides were then washed and mounted in a glycerol-based mounting medium containing Gelvatol and 2.5%
1,4-diazabicyclo[2.2.2]octane (Sigma).
Cell Lysates and Immunoprecipitation--
After the first day in
culture medium containing 10% serum, cardiomyocytes were washed and
cultured in minimal medium (4:1 Dulbecco's modified Eagle's
medium/medium 199; 100 units/ml penicillin, 100 µg/ml streptomycin,
and 10 mM glutamine) and then treated with NRG-1 and/or
IGF-I for 5 min. Cells extracts were obtained by applying cold lysis
buffer (0.7 ml/10 cm plate for 10 min) containing 20 mM
imidazole, pH 6.8, 100 mM KCl, 2 mM
MgCl2, 10 mM EGTA, 300 mM sucrose,
1 mM sodium vanadate, 1 mM sodium molybdate, 1 mM sodium fluoride, 0.2% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride in PBS. All subsequent
steps were performed at 4 °C as described previously (29). Briefly,
cell lysates were precleared with 200 µl/ml 10% protein A-Sepharose
in 1% ovalbumin. Affinity purified erbB2 antibodies (Santa Cruz, CA)
were added to 0.3 ml of lysate at a concentration of 1 µg/ml and 50 µl/ml of 10% protein A-Sepharose (Amersham Pharmacia Biotech) in 1%
ovalbumin for 1 h. Alternatively, 10 µl of rabbit anti-erbB4
serum raised against recombinant protein (generous gift of Dr. Cary
Lai, Scripps Research Institute, La Jolla, CA) was added to 0.3 ml of
cell lysate. For control immunoprecipitates, preimmune serum was added
to 0.3 ml of cell lysate. The protein precipitates were washed four
times with lysis buffer, and proteins were analyzed by 6.5%
SDS-polyacrylamide gel electrophoresis. Immunoblotting was performed
with anti phosphotyrosine antibodies (RC20H, Transduction
Laboratories), erbB2, or erbB4 antibodies.
Analysis of NRG-1 Function on the Developing Cardiac Chamber in
Cultured Whole Mouse Embryos--
We have chosen to analyze the
effects of injected growth factors on cardiac development from E11.5 to
E12.5 (Fig. 1, A and B), when active trabeculation of the ventricular wall and
expansion of the cushions is occurring (30). The diffusion of peptide growth factors throughout the heart following direct cardiac
administration was analyzed by injection of the fluorescent markers,
DiI (Molecular Probes, Eugene, OR) or fluorescein
isothiocyanate-dextran. The hydrophobicity of DiI allows for its
cellular uptake, whereas the molecular weight of fluorescein
isothiocyanate-dextran is in the range of NRG-1 or IGF-I peptides.
Following intracardiac injection, DiI or fluorescein
isothiocyanate-dextran diffused in both the right and left ventricular
chambers and to the atria and conotruncus, suggesting that the
injection protocol will effectively deliver growth factors to the
entire heart. The distribution of DiI was stable after 24 h in
culture (Fig. 1, C and D).
The biologically active recombinant peptide containing the epidermal
growth factor-like domain of NRG- and IGF-I-induced Expansion of Atrioventricular Cushion
Tissue--
The NRG-1 expression pattern indicates a potential role in
cushion mesenchyme development. Therefore, the activity of NRG-1 during
expansion of AV cushion tissue after intracardiac injection of NRG-1
was analyzed by scanning electron microscopy. The three-dimensional morphology of the developing atrioventricular cushions is shown in Fig.
3. We used the physical distance between
the superior and inferior AV cushions as an indicator of AV mesenchymal
expansion. The atria have been removed for ease of visualization of
cushion tissue. Accordingly, representative dorsal views of the
atrioventricular cushions in E11.5 mouse embryos after 24 h in
culture are shown in Fig. 3 and summarized in Table I. As shown in
Table I, NRG-1 induced a consistent growth of cushion mesenchyme,
although it was not statistically significant. The distance between the
two AV cushions was reduced in response to NRG-1 to 33 ± 13 µm
(Fig. 3B) compared with 48 ± 12 µm in control
PBS-injected embryos (Fig. 3A). This slight cushion growth
in response to NRG-1 suggested that additional interacting signals were
required for cushion development. Accordingly, we examined whether
another growth factor, IGF-I, which is expressed in cushion mesenchyme
(10), may potentiate the NRG-1 effect on cushion morphogenesis because
in vitro work by others has demonstrated a functional
interaction between the erbB receptor family and IGF-I receptor signal
pathways (35, 36). Injection of IGF-I alone into the heart did not
induce expansion of cushion mesenchyme (Table I and Fig.
3C). However, the combination of both IGF-I and NRG-1
consistently reduced the AV cushion distance to an average of 22 ± 2 µm (Fig. 3D), suggesting a synergistic effect on
expansion of cushion mesenchyme in the developing heart of treated
embryos in culture. In addition, the combined injection of NRG-1 and
IGF-I induced partial fusion of the cushions, which occurred in 50% of
treated embryos. In normal development in vivo, comparable
fusion of the cushions is not observed prior to E13 (30).
NRG-1 and IGF-I Synergistically Promote Compact Zone Expansion and
Activates Ventricular Muscle Cell Proliferation--
The
three-dimensional morphology of the ventricular chamber was examined
using scanning electron microscopy on the parietal and septal sections
of the developing heart. From these images, the depth of the compact
zone of the free wall of the ventricles can be determined. Measurements
of the thickness of the compact zone were acquired at a location of the
right ventricular free wall immediately dorsal to the apex of the
chamber. The average distance of the ventricular compact zone was
16 ± 1 µm (1-2 cell layers) in untreated embryos (Fig.
4A) and was not modified by NRG-1 or IGF-I injections (Fig. 4, B and C). A
remarkable effect on the ventricular compact zone was observed by
combined injections with both IGF-I and NRG-1. The two growth factors
together consistently expanded the compact zone to an average of
26 ± 2 µm (Fig. 4D), which represents an increase to
3-5 layers.
The morphological changes induced by combined injection of NRG-1 and
IGF-I may be due in part to stimulation of proliferation of the
respective cardiac lineages in the ventricular chambers and endocardial
cushion tissue (ventricular myocytes and endocardial-derived mesenchyme). Therefore, we analyzed proliferation by monitoring nuclear
incorporation of exogenous BrdUrd. No differences in cardiomyocyte BrdUrd incorporation were observed between control, PBS-injected, NRG-1-injected, or IGF-I-injected embryos (Fig.
5, A-C). Combined NRG-1/IGF-I
injections in developing embryos resulted in active BrdUrd
incorporation in 56 ± 6% of trabecular cardiomyocytes (Fig. 5D) versus 24 ± 4% of BrdUrd positive
cells in trabeculae of control or single factor-injected embryos (Fig.
5, A-C). Therefore, the novel observation on the expansion
of the compact zone (Fig. 5D) induced by combined
NRG-1/IGF-I injection was accompanied by an increased DNA synthesis as
assessed by BrdUrd incorporation (Fig. 5D,
arrows). Differentiation of cardiac chambers, as evidenced by proper down-regulation of the atrial form of myosin light chain, MLC-2a in ventricular cardiomyocytes (37), occurred normally in NRG-1-
and IGF-I-injected embryos (data not shown). Collectively, these
results indicate that the potentiation of NRG-1 and IGF-I activities
increase the ability of cardiomyocytes to proliferate and differentiate
in developing embryos.
Concerted Activation of Cultured Cardiomyocyte Growth by NRG-1 and
IGF-I--
The analysis of the function of NRG-1 and IGF-I on cardiac
muscle cells in the absence of additional effects from nonmuscle cells
was carried out in isolated neonatal rat cardiomyocytes, which are
readily purified from other cells present in the heart. The specific
nuclear incorporation of BrdUrd in cardiomyocytes, an index of DNA
synthesis, was determined subsequent to treatment with NRG-1 and/or
IGF-I. As shown in Fig. 6A,
the low DNA synthesis rate in cardiomyocytes in culture is
increased in serum-containing medium. Significantly higher stimulation
of BrdUrd incorporation was achieved in cardiomyocytes grown in the
combined presence of NRG-1 and IGF-I versus untreated or
NRG-1 or IGF-I or serum-stimulated cells (Fig. 6A). The
effect of combined NRG-1/IGF-I on BrdUrd incorporation was prevented by
wortmannin, a specific inhibitor of the PI 3-kinase (Fig.
6A). Changes in DNA synthesis were quantified by BrdUrd
incorporation as well as [3H]thymidine uptake in cell
lysates (Fig. 6B). A 2-fold stimulation of DNA synthesis was
observed in cardiomyocytes treated with NRG-1 or IGF-I alone. The level
of [3H]thymidine incorporation observed in cardiomyocytes
treated with NRG-1 and IGF-I was increased by 10-fold versus
untreated cardiomyocytes, representing a higher incorporation
compared with the 6-fold induction observed in serum-stimulated
cardiomyocytes. The level of DNA synthesis induced by combined NRG-1
and IGF-I was consistent with a synergistic response of cardiomyocytes
to both growth factors. Two major signaling pathways that are triggered
by the activation of either erbB2 or IGF-I receptors are the MAP kinase
and the PI 3-kinase pathways (38-40). Therefore, to dissect the
cascade of events triggered by the combined treatment of NRG-1 and
IGF-I, we used wortmannin, an inhibitor of PI 3-kinase and the MAP
kinase, mitogen-activated extracellular-signal regulated kinase
specific inhibitor PD098059 and by assessing the effect of dominant
negative molecules of either MAP or PI 3-kinase pathways.
NRG-1/IGF-I-stimulated DNA synthesis was blocked by addition of
wortmannin (Fig. 6B), whereas it was not prevented by
PD098059, which blocks the response of cardiomyocytes to the individual
effects of NRG-1 and IGF-I (Fig. 6B).
Adenoviral infection of cardiomyocytes mediated highly efficient
delivery of dominant negative mutants of the MAP kinase pathway (Ras,
and Raf) or of the PI 3-kinase pathway (Rac) (data not shown). NRG-1/IGF-I induced BrdUrd incorporation to an average of 18 ± 3% positive cells/100 cells and was significantly inhibited to basal
levels by dominant negative Rac1 (
Concordant with previous observations (41), a rapid phosphorylation of
erbB2 and erbB4 receptors was induced by NRG-1 in treated
cardiomyocytes in culture (Fig.
7A). In the presence of NRG-1,
the cardiomyocytes showed an elongated shape with an increased and
highly organized sarcomere structure (Fig. 7B). Although
IGF-I has been shown to induce IGF-I receptor phosphorylation (42), it
had no apparent effect on cardiomyocyte growth and morphology. The
presence of both NRG-1 and IGF-I dramatically induced cardiomyocyte growth, which acquired a more organized myofilament structure (Fig.
7B), indicative of an effect to promote differentiation. Although NRG-1-stimulated myofibrillogenesis was abolished by the MAP
kinase inhibitor, PD098059, or the PI 3-kinase inhibitor wortmannin,
the combined effect of NRG-1/IGF-I was only prevented by wortmannin
(data not shown). To determine whether biochemical interactions between
receptors could be involved in the NRG-1/IGF-I-mediated PI 3-kinase
activation, the phosphorylation level of the erbB receptors was assayed
in the presence of NRG-1 or IGF-I, because this is the earliest event
occurring upon activation. As shown in Fig. 7C, erbB2
phosphorylation was detected in the presence of NRG-1 but not IGF-I,
suggesting that there is no cross-activation of the erbB2 receptor by
IGF-I. However, a weak signal corresponding to the The Whole Mouse Embryo Culture System Allows Identification of
Potential Paracrine Signals for Cardiac Morphogenesis in Intact
Embryos--
A growing body of evidence suggests that muscle cell
nonautonomous mechanisms mediated by paracrine factors may play a
critical role in cardiac chamber morphogenesis (43, 44). Therefore, an
ideal system for assaying the effects of potential growth factors in
the heart would be one that allows for subsequent cardiac development to take place in the intact embryo where cell-cell communication is
maintained. As has been previously noted, mouse embryos are able to
predictably develop in culture and even respond to grafted cells (19,
45). The intracardiac delivery of peptide growth factors combined with
the ability to monitor the maturation of specific cardiac compartments
(conotruncal ridges, AV cushions, trabeculae, and compact zone)
enables the identification and functional relevance of secreted
molecules that act on different cardiac cell populations. In addition,
this system will be a valuable tool in performing rescue studies of
mutant phenotypes following delivery of defined factors.
NRG-1 and IGF-I Act Synergistically on Cardiac Cushion Tissue
Formation--
Previous studies utilizing in vitro culture
explants of the chicken atrioventricular region have established a
paradigm in which signals that originate in the AV myocardium are
required to trigger competent endothelial cells from the AV endocardium to form cushion mesenchyme (5). Through direct injection into the
intact embryo, we now provide evidence that instructive signals for
expansion of cushion mesenchyme reside in cells of the endocardium. Thus, two factors, NRG-1 and IGF-I, whose expression is localized to
endocardial and mesenchymal cells, respectively, can act
synergistically to promote expansion of cushion mesenchyme. The
consequent AV endocardial cushion growth was quantified by proximity of
the superior and inferior cushions to each other via scanning electron microscopy. In some cases the two cushions appeared fused and indicated
a precocious expansion comparable with cushion growth normally observed
at E13 (30). Taken together, these results constitute a novel
demonstration of a role for NRG-1 and IGF-I in cushion morphogenesis
and maturation.
In the cushions, the effect of endocardial-derived NRG-1 may be
mediated through erbB3 receptors present in mesenchymal cells adjacent
to the endocardial cells (7, 13). In these studies, we have detected
expression of erbB4 in the AV cushions at later stages of embryo
development, E13.5 (data not shown). It will be intriguing to examine
whether erbB4 is indeed the erbB3 partner in cushion mesenchyme because
the later can bind NRG-1 but lacks tyrosine kinase activity (15).
NRG-1 and IGF-I Synergistically Promote Compact Zone Expansion and
Activate Ventricular Muscle Cell Proliferation--
In intact embryos
in culture, the effect of exogenous NRG-1 was manifested by the
appearance of organized projections of the ventricular wall, which were
marked by the expression of AchE. In agreement with a requirement of
NRG-1 for trabeculation, our results indicate that NRG-1 induces the
highly proliferative cardiomyocytes of the compact zone down a
differentiation pathway, thereby promoting the transition of compact
zone myocardium into trabecular muscle without modification of basal
levels of DNA synthesis.
A major transformation of the ventricular wall induced by the effect of
combined NRG-1/IGF-I was the expansion of the compact zone, an event
normally occurring after E13 (30). Previous work has indicated that
IGF-I is involved in myofibrillogenesis of ventricular cardiomyocytes
because inactivation of the IGF-I gene resulted in cardiomyocytes with
reduced sarcomeric structures. A proliferative effect of IGF-I has been
shown in transgenic mice overexpressing IGF-I in the mature heart (46).
However, it had no apparent effect on cardiac development. The present
study documents that the interaction of NRG-1 and IGF-I signaling
pathways has a synergistic effect on cushion tissue and ventricular
cardiomyocyte growth and maturation. The induced increase in DNA
synthesis in ventricular cardiomyocytes was accompanied by normal
differentiation, as assessed by down-regulation of MLC-2a. In this
regard, proliferation and differentiation can simultaneously occur in
cardiomyocytes, as opposed to skeletal muscle cells where these
processes appear to be mutually exclusive (47, 48). Indeed, the
synergistic effect of combined NRG-1 and IGF-I dramatically increased
both DNA synthesis and myofibril organization in cultured
cardiomyocytes, which indicates the ability of cardiomyocytes to both
proliferate and differentiate.
Concerted Activation of PI 3-Kinase Mediated by NRG-1 and
IGF-I--
Early events of NRG-1- or IGF-I-mediated cellular responses
involves the phosphorylation of their cognate receptors inducing recruitment of SH2 domain adapter molecules that belong to the MAP
kinase or PI 3-kinase cascades (38-40, 48, 49). In the present study,
the combined effect of NRG-1 and IGF-I appeared to converge on the
activation of the PI 3-kinase and was reflected in a significant
stimulation of DNA synthesis and differentiation both in cultured
cardiomyocytes and in the intact heart in whole embryo culture. The
synergistic effect of NRG-1 and IGF-I supports the concept that
interplaying signals are required for complete induction of a given
phenotypic change (50, 51). The accumulated evidence indicates that
transactivation of members of the erbB receptor family may occur by
direct interaction or erbB2-mediated lateral signal transmission (52,
53). Recently, association of gp130 subunits of the interleukin-6
receptor to erbB2 phosphorylation was observed in an
interleukin-6-treated carcinoma cell line (54). In addition, activation
of the epidermal growth factor receptor mediates intracellular
transactivation of the IGF-I receptor and reconstitutes the
poliferative and transforming ability of certain IGF-I receptor
mutations (36).
It has been shown that either NRG-1 or IGF-I induces convergent
signaling through interaction of Ras-MAP kinase and PI 3-kinase pathways, which mediate inducible gene expression (55) or protection from apoptosis (56). In our studies, phosphorylation of the erbB
receptors in cardiomyocytes was mediated by NRG-1 alone but not by
IGF-I alone, suggesting that there is no cross-activation of the erbB
receptors by IGF-I. This may indicate that the presence of both NRG-1
and IGF-I may be required downstream of phosphorylation of erbB2 such
as the p85 regulatory subunit of PI 3-kinase. Indeed, after 5 min of
NRG-1/IGF-I treatment, erbB2 receptors co-immunoprecipitated with the
p85 regulatory subunit of PI 3-kinase (data not shown), suggesting that
the biochemical interaction of erbB2 receptors with p85 may involve
additional phosphorylation of erbB2 tyrosine residues mediated by
combined NRG-1/IGF-I that was below the sensitivity of our
phosphorylation assay. Alternatively, the NRG-1/IGF-I-induced phenotypic changes could be mediated by additional growth factors that
could, in turn, activate PI 3-kinase.
The contribution of Ras to the NRG-1/IGF-I-mediated effects on
cardiomyocytes may occur through convergent activation of PI 3-kinase
or may directly affect the cellular response. This latter event would
appear to be unlikely because inhibition of mitogen-activated extracellular-signal regulated kinase did not modify the cardiomyocyte response to NRG-1/IGF-I. In this regard, the partial inhibition mediated by dominant negative Ras and the complete block mediated by
either dominant negative Rac1 or wortmannin treatment support the
concept that convergence of the NRG-1 and IGF-I signaling pathways
occurs at the level of PI 3-kinase pathway activation. In
wortmannin-injected embryos, preliminary results have shown a slight
reduction of the basal level and an inhibition of the combined
NRG-1/IGF-I-mediated stimulation of cardiomyocyte-incorporated BrdUrd,
indicating that PI 3-kinase is indeed required for cardiomyocyte proliferation in the intact embryo. In this regard, it will be of
interest to determine the morphologic effects of inhibiting PI 3-kinase
using wortmannin and dominant negative Rac1 during normal cardiogenesis
in whole mouse culture. Additionally, it will be of interest to
examine, in detail, the requirement of the PI 3-kinase during cell
proliferation and differentiation that ultimately results in formation
and remodeling of cardiac structures during development. It is also
intriguing to examine whether the synergistic effect of NRG-1 and IGF-I
mediated by activation of PI 3-kinase plays a role in the balance
between cell proliferation, survival, and morphological cellular
changes during the stress-activated hypertrophic response in the mature heart (57).
We thank Dr. Cary Lai for fruitful
discussions and cDNA probes, Dr. Mark Sliwkowski for recombinant
heregulin *
This work was supported by grants from the National
Institutes of Health (to K. R. C.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The abbreviations used are:
NRG-1, neuregulin-1;
IGF-1, insulin-like growth factor-1;
En, embryonic day
n post-coitum;
MAP, mitogen-activated protein;
PI, phosphatidylinositol;
PBS, phosphate-buffered saline;
AchE, acetylcholinesterase;
BrdUrd, bromodeoxyuridine;
GFP, green fluorescent
protein;
Av, atrioventricular;
DiI, chloromethylbenzamido octadecyl
indocarbocyanine.
Synergistic Roles of Neuregulin-1 and Insulin-like Growth
Factor-I in Activation of the Phosphatidylinositol 3-Kinase Pathway and
Cardiac Chamber Morphogenesis*
,
University of California, San Diego/Salk
Program in Molecular Medicine, Department of Medicine and Center for
Molecular Genetics, University of California, San Diego, La Jolla,
California 92093-0613, the § Department of Cell Biology and
Anatomy, Medical University of South Carolina, Charleston, South
Carolina 29425-2204, and the ¶ Department of Physiology,
University of Maryland, Baltimore, Maryland 20212
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
form (21) (Genentech, Inc., San Francisco, CA) or
form (gift from Kuo-Fen Lee, Salk Institute, La Jolla, CA) were 50 ng/µl, and IGF-I was 50 ng/µl (Genentech, Inc.). These
concentrations were selected based on dose-response curves performed in
cultured cardiomyocytes and adjusted in the embryo culture for
NRG-1.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Development of murine embryos in
culture. A, mouse embryos were dissected at E11.5 and
placed in culture. B, following 26 h, which corresponds
to stage E12.5 as determined by the limb development
(arrow), the embryos were removed and perfusion fixed either
for paraffin embedding or for scanning electron microscopy.
C and D illustrate the range of diffusion and
cellular uptake of 1 µl of DiI in the cardiac chambers following a
single injection into the left ventricle. Incorporation of DiI was
found in the conotruncus (CT), the right (RV) and
left (LV) ventricles, and the left atria
(LA).
isoform of NRG-1 was injected into the
ventricular chamber of wild type embryos at E11.5 and allowed to
develop in culture to E12.5. As trabeculation of the ventricular wall
may depend on NRG-1, expression of acetylcholinesterase (AchE), whose
enzymatic activity in the embryonic rat heart is restricted to
cardiomyocytes of the trabeculae of the ventricular wall, was selected
to follow induced trabecular growth (31, 32). Accordingly, we observed
AchE mRNA in a restricted expression pattern coincident with the
trabeculae and in continuity with the conotruncus at early stages of
in utero mouse embryo development, E10-E13 (data not
shown). To monitor expansion of trabeculae in NRG-1 or control
PBS-injected embryos in culture, the expression of AchE was determined
by in situ hybridization in comparative histological
sections of the ventricular free wall. A representation of the
corresponding ventricular area used for the analysis is shown in Fig.
2A. As observed by the number
of projections of cardiomyocytes forming trabeculae marked by AchE
expression, NRG-1-injected embryos display an increased density of
trabeculae (Fig. 2C) compared with control injected embryos
(Fig. 2B). Ventricular trabeculae growth in NRG-1-injected
embryos was accompanied by a 1.5-fold increase in the number of
cardiomyocytes (Table I). Similar effects were observed with NRG-1 recombinant peptides of either or isoforms (data not shown). Intracardiac injection of IGF-I, a peptide
growth factor not related to NRG-1, had no significant effect on
trabeculation. The AchE expression pattern in IGF-I-injected embryos
was not different from control PBS-injected embryos (Fig. 2D). These results indicate that exogenous NRG-1 induced the
differentiation of the highly proliferative cardiomyocytes in the
ventricular compact zone into trabeculae, in agreement with previous
work on the requirement of NRG-1 for the formation of trabeculae (7, 33, 34).
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Fig. 2.
AchE mRNA expression in NRG-1-injected
embryos. Sagittal sections of treated E12.5 embryos were
hybridized with riboprobes for AchE. A, sections of the free
wall ventricle were chosen to examine trabeculae separate from the
interventricular septum and atrioventricular cushions. Heart sections
of control PBS-injected (B), NRG-1-injected (C),
and IGF-I-injected embryos (D). NRG-1 stimulated trabecular
growth is marked by increased AchE expression.
Effect of injected growth factors on cardiac development in cultured
embryos
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Fig. 3.
Effect of treatment with NRG-1 and IGF-I on
expansion of the superior and inferior atrioventricular cushions as
assessed by scanning electron microscopy. The orientation in each
panel is with the superior cushion pad on the left and
inferior cushion on the right. A, PBS control.
B, NRG-1. C, IGF-I. D, NRG-1 plus
IGF-I. Treatment with both growth factors resulted in a consistent
expansion of the AV cushions that was the not seen with either factor
alone. All images were acquired from the top of the ventricular chamber
at a primary magnification of 160×.
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Fig. 4.
Effect of treatment with NRG-1 and IGF-I on
expansion of the compact zone. Embryos were injected with 1 µl
of PBS (A), 50 ng/µl RG-1 (B), 50 ng/µl IGF-I
(C), or NRG-1 and IGF-I (D) and then processed
for scanning electron microscopy. Note the expansion of the compact
zone occurred only in the presence of the combination of NRG-1 and
IGF-I. All images were acquired from the right ventricular free wall at
a primary magnification of 1000×. The region in brackets
denotes the thickness of the compact zone.
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Fig. 5.
Determination of DNA synthesis in
cardiomyocytes during whole embryo culture as assessed by BrdUrd
incorporation in vivo. Peroxidase reaction was performed in
sagittal sections of E12.5 embryos developed in culture following
immunostaining with peroxidase-coupled antibodies against BrdUrd and
counterstained with hematoxylin. A, control injected
embryos. The arrowhead identifies a representative BrdUrd
positive nuclei in cardiomyocytes of compact zone and endocardial
cells. B, NRG-1-injected embryos. There was an increase in
the total number of cells in the trabecular region of the ventricular
free wall as described under "Materials and Methods" with no
significant increase in the relative number of BrdUrd positive cells.
C, IGF-I-injected embryos. The relative number of BrdUrd
positive cells are comparable with control sections (see A).
D, NRG-1/IGF-I-injected embryos. There was an increased
relative number of BrdUrd positive cardiomyocytes in both trabeculae
and compact zone in combined NRG-1- and IGF-I-injected embryos.
Arrows identify BrdUrd positive nuclei in trabeculae and in
the expanded compact zone. Combined NRG-1/IGF-I treatment also induced
expansion of the ventricular compact zone.
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Fig. 6.
DNA synthesis in cultured
cardiomyocytes. Cardiomyocytes were cultured in serum-free medium
(M), or in the presence of 10% serum (S), 2.5 nM NRG-1 (N), 5 nM IGF-I
(I), or NRG-1 plus IGF-I (IN). Alternatively,
cardiomyocytes were treated with 1 µM wortmannin
(W), an inhibitor of PI 3-kinase or 10 µM
PD098059 (PD), an inhibitor of MAP kinase before addition of
growth factors. A, representative images of cardiomyocytes
immunostained for BrdUrd incorporation. Note that a higher number of
cells are BrdUrd-positive in the combined presence of NRG-1 and IGF-I
and that this increase is blocked by PI 3-kinase inhibitor, wortmannin.
B, quantitation of BrdUrd or [3H-]thymidine
incorporation demonstrated that the synergistic effect of NRG-1/IGF-I
on proliferation can be blocked by wortmannin but not the MAP kinase
inhibitor, PD098059 (n = 6 individual experiments).
C, representative images of myocytes immunostained for
BrdUrd incorporation after treatment with NRG-1/IGF-I in the absence
(IN) or presence of adenoviral clones containing dominant
negative Rac1 ( 
Rac, 50 particles/cell) or GFP or containing
activated Rac1 (Rac*, 100 particles/cell) in the absence of growth
factors. Note the inhibitory effect of Rac on BrdUrd incorporation.
The control virus containing GFP (100 particles/cell) had no effect on
BrdUrd incorporation. D, quantification of BrdUrd
incorporation illustrated that the effect of NRG-1/IGF-I is completely
abolished by dominant negative Rac1 (Rac) and only partially
inhibited by dominant negative Ras (Ras) or dominant negative Raf
(Raf). Rac* alone, but not Raf*, had an effect to increase BrdUrd
incorporation (n = 4 individual experiments). *,
p < 0.001.
Rac, 1 ± 0.4%) and
partially inhibited by dominant negative Ras (Ras, 7.5 ± 1%)
but not by dominant negative Raf (Raf) (Fig. 6, C and
D). Because Rac1/Akt is a downstream mediator of activated
PI 3-kinase, this result confirms the requirement of PI 3-kinase for
induced BrdUrd incorporation, an index of DNA synthesis. In addition,
BrdUrd incorporation was partially induced by an activated form of Rac1
(Rac*) to an average of 12 ± 2% versus 1 ± 0.5% in control cardiomyocytes, which were infected with adenovirus
expressing GFP (Fig. 6C) or an activated form of Raf (Raf*)
(data not shown).
and forms of
the p85 regulatory subunit of the PI 3-kinase was only detected in
erbB2 immunoprecipitates and peaked at 10 min of NRG-1/IGF-I treatment
(data not shown). In this regard, a direct interaction with PI 3-kinase
may require phosphorylation of additional tyrosine residues of
NRG-1-activated erbB2 receptor in the presence of IGF-I. The activated
Rac1 mutant could induce DNA synthesis to a comparable, however not
identical, level of BrdUrd incorporation observed in combined
NRG-1/IGF-I-treated cardiomyocytes. In addition, Ras activation appears
to be involved in the cellular phenotypic changes induced by combined
NRG-1/IGF-I treatment, which could occur via a convergence on
activation of PI 3-kinase and unlikely to occur through MAP kinase
because inhibition of mitogen-activated extracellular-signal regulated
kinase did not modify the cardiomyocyte response to NRG-1/IGF-I. Based
on these results, the synergistic effect of NRG-1 and IGF-I is likely mediated through convergence of signaling cascades via a direct- and
Ras-mediated activation of PI 3-kinase.
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Fig. 7.
Detection of early activation of the erbB
receptors and morphological changes induced by NRG-1 and NRG-1/IGF-I in
cardiomyocytes. A, tyrosine phosphorylation of erbB2
and erbB4 receptors. Lysates of cardiomyocytes grown in serum-free
medium or with NRG-1 for 4 min were subjected to immunoprecipitation
(IP) with erbB2 affinity purified antibodies or erbB4
antiserum. Phosphorylation was detected in immunoblots with RC20H
phosphotyrosine monoclonal antibodies. B, cardiomyocytes
were grown in serum-free medium (Co), or in the presence of
NRG-1 (N), IGF-I (I), or NRG-1/IGF
(IN) for 72 h and immunostained for myomesin
(red) or DAPI (blue). C, tyrosine
phosphorylation of the erbB2 receptor was assayed in lysates of
cardiomyocytes treated with NRG-1, IGF-I, or both.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
, Dr. Kuo-Fen Lee for recombinant neuregulin-1 , Dr.
David Luo for acetylcholinesterase cDNA probe, Dr. Robert Price for
sharing the scanning electron microscope, and Dr. Tristan Bahnson for
use of the Pipette puller. We are indebted to Mahmoud Itani and Janelle
Stricker for skillful technical assistance and Julie Anderson for
maintaining the mouse colony and specially grateful to Dr. Pilar
Ruiz-Lozano for practical advice and Dr. Sylvia Evans for insightful
discussions and critical review of the manuscript.
FOOTNOTES
To whom correspondence should be addressed: Dept. of Medicine,
0613-C, University of California, San Diego, 9500 Gilman Dr., La Jolla,
CA 92093-0613. Tel.: 619-534-6835; Fax: 619-534-8180; E-mail:
kchien@ucsd.edu.
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