From the Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115 and ¶ Cambridge NeuroScience Inc., Cambridge, Massachusetts 02139
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ABSTRACT |
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Neuregulins (i.e. neuregulin-1
(NRG1), also called neu differentiation factor, heregulin,
glial growth factor, and acetylcholine receptor-inducing
activity) are known to induce growth and differentiation of
epithelial, glial, neuronal, and skeletal muscle cells. Unexpectedly, mice with loss of function mutations of NRG1 or of either of two of
their cognate receptors, ErbB2 and ErbB4, die during midembryogenesis due to the aborted development of myocardial trabeculae in ventricular muscle. To examine the role of NRG and their receptors in developing and postnatal myocardium, we studied the ability of a soluble NRG1
(recombinant human glial growth factor 2) to promote proliferation, survival, and growth of isolated neonatal and adult rat cardiac myocytes. Both ErbB2 and ErbB4 receptors were found to be expressed by
neonatal and adult ventricular myocytes and activated by rhGGF2. rhGGF2
(30 ng/ml) provoked an approximate 2-fold increase in embryonic cardiac
myocyte proliferation. rhGGF2 also promoted survival and inhibited
apoptosis of subconfluent, serum-deprived myocyte primary cultures and
also induced hypertrophic growth in both neonatal and adult ventricular
myocytes, which was accompanied by enhanced expression of prepro-atrial
natriuretic factor and skeletal -actin. Moreover, NRG1 mRNA
could be detected in coronary microvascular endothelial cell primary
cultures prepared from adult rat ventricular muscle. NRG1 expression in
these cells was increased by endothelin-1, another locally acting
cardiotropic peptide within the heart. The persistent expression of
both a neuregulin and its cognate receptors in the postnatal and adult
heart suggests a continuing role for neuregulins in the myocardial
adaption to physiologic stress or injury.
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INTRODUCTION |
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An increasing number of locally acting biologic mediators have now been implicated in regulating myocardial development as well as the subsequent adaptive growth response of postnatal cardiac muscle to physiologic stress. Aside from acetylcholine and the biogenic amines, most of these mediators are locally acting peptide growth factors that are synthesized by cardiac muscle cells themselves or by other parenchymal cells within the heart, such as endothelial cells of the microvasculature and endocardium (1-6). Endothelins and angiotensins, for example, both of which have been shown to induce a hypertrophic growth in cardiac myocytes, are synthesized and released by endothelial cells of the coronary microvasculature (7-10).
Another growth factor signaling system that has recently been
implicated in cardiac development is the "neuregulins," a family of
locally acting peptide autocoids known to be important in the development of the central and peripheral nervous system. Targeted disruption of the neuregulin-1
(NRG1)1 gene or of either of
two neuregulin receptors (ErbB2 or ErbB4) led unexpectedly to death
during midembryogenesis from aborted development of trabeculation in
ventricular muscle of the fetal myocardium (11-15). These reports
indicated that neuregulins, released from the endocardial endothelium,
are essential for the growth and phenotypic adaptation of subjacent
cardiac myocytes during development. Neuregulins include the
growth-regulatory proteins: glial growth factor (GGF), Neu
differentiation factor, heregulin, and acetylcholine receptor-inducing
activity. All of these proteins are encoded by a single gene but exist
in at least 15 isoforms, including both integral membrane and soluble
signaling proteins (16). Recently, two additional genes coding for
neuregulin-like signaling proteins have been identified that have been
termed "neuregulin-2" (NRG2), and "neuregulin-3" (NRG3)
(17-19). Products of the originally identified neuregulin family, now
termed neuregulin-1 (NRG1), share about 40 and 20% amino acid sequence
identity with NRG2 and NRG3, respectively. Unlike NRG1-derived
mRNAs, which within the developing heart are limited largely to the
endocardial endothelium of ventricular muscle, NRG2 mRNAs are found
primarily in the endothelium of developing atrial muscle, while no NRG3 mRNA has been detected by in situ hybridization in the
heart at midembryogenesis (18, 19). Neuregulins, which are produced by
either neuronal or mesenchymal cells, mediate their effects by binding
to and signaling via the ErbB family of receptors, including
ErbB1/epidermal growth factor receptor, Neu/ErbB2/HER2, ErbB3/HER3, and
ErbB4/HER4 (16, 20). All neuregulins (i.e. NRG1, NRG2, and
NRG3) identified to date bind to either ErbB3 or ErbB4, subsequently
recruiting another ErbB receptor (including ErbB1 or ErbB2) as
co-receptors to initiate signaling. While all possible hetero- and
homodimers of the ErbB receptor family can be formed, there is no known
high affinity ligand for ErbB2, and ErbB3 homodimers have diminished
intrinsic tyrosine kinase activity, suggesting that there is a limited
and hierarchical structure to neuregulin/ErbB signaling in target cells
(20). In the case of myocardial development, for example, animals
lacking functional ErbB2 (ErbB2/
) exhibited a cardiac phenotype
similar to that of animals with a targeted disruption of the NRG1 gene,
indicating that ErbB4 homodimers could not substitute for ErbB2 in
fetal ventricular myocytes at this point in development (13). Peptide ligands other than products of the known neuregulin genes may also
initiate ErbB signaling. Heparin-binding epidermal growth factor-like
growth factor, which we have demonstrated can act as an autocrine
growth stimulus in both neonatal and adult ventricular myocytes (21),
has been shown also to act as a ligand for both the ErbB1 and ErbB4
receptors (22). Indeed, as noted by Burden and Yarden (20), this
abundance of potential ligands suggests that the spatial and temporal
restriction of soluble and membrane-bound neuregulins and other
ErbB-receptor ligands provides the necessary specificity for
ErbB-dependent signaling.
Neuregulins have been shown to regulate the proliferation, differentiation, and survival of Schwann cells and oligodendrocytes (23-26), neurite extension of retinal neurons (27), the maturation of skeletal muscle myoblasts (28), and expression of acetylcholine receptors in skeletal muscle (29, 30). However, direct evidence for effects of neuregulins on cardiac myocytes has not been reported. Here, we examine the ErbB receptor expression in neonatal and adult rat ventricular myocytes. We demonstrate that a soluble recombinant form of a human NRG1, glial growth factor 2 (rhGGF2), promotes embryonic cardiac myocyte proliferation and the growth and survival of both neonatal and adult ventricular myocytes in vitro. We also examine the expression of NRG1 in primary cultures of coronary microvascular endothelial cells These data support the concept that the neuregulin-ErbB signaling system plays an important role in the onset of myocardial trabeculation and cardiac morphogenesis by promoting the proliferation, survival, and maturation of cardiomyocytes and suggest that this system may remain an important pathway for phenotypic adaptation in both neonatal and adult myocardium.
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EXPERIMENTAL PROCEDURES |
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Preparation of Cardiac Myocyte and Non-myocyte Primary Cultures-- Embryonic and neonatal rat ventricular myocyte (NRVM) primary cultures were prepared as described previously (31). To selectively enrich for myocytes, dissociated cells were centrifuged twice at 500 rpm for 5 min, preplated twice for 75 min, and finally plated at low density (0.7-1 × 104 cells/cm2) in Dulbecco's modified Eagle's medium (DMEM; Life Technologies Inc.) supplemented with 7% fetal bovine serum (FBS; Sigma). Cytosine arabinoside (10 µM; Sigma) was added during the first 24-48 h to prevent proliferation of non-myocytes, with the exception of cultures used for thymidine uptake measurements. Unless otherwise stated, all experiments were performed 36-48 h after changing to a serum-free medium, DMEM plus ITS (Sigma). Using this method, we routinely obtained primary cultures with >95% myocytes, as assessed by microscopic observation of spontaneous contraction and by immunofluorescence staining with a monoclonal anticardiac myosin heavy chain antibody (anti-MHC; Biogenesis, Sandown, NH).
Primary cultures of cellular fractions isolated from neonatal hearts enriched in non-myocyte cells were prepared by passaging twice cells that adhered to the tissue culture dish during the preplating procedure. These non-myocyte cultures, which contained few anti-MHC positive cells, were allowed to grow to subconfluence in DMEM supplemented with 20% FBS before switching to DMEM plus ITS for a subsequent 36-48 h. Isolation and preparation of adult rat ventricular myocyte (ARVM) primary cultures was carried out using techniques previously described (32). Rod-shaped cardiac myocytes were plated in culture medium on laminin- (10 µg/ml; Collaborative Research, Bedford, MA) precoated dishes for 60 min, followed by one change of medium to remove loosely attached cells. The contamination of ARVM primary cultures by non-myocytes was determined by counting with a hemocytometer and was typically less than 5%. All ARVM primary cultures were maintained in a defined medium termed "ACCITT" (32) composed of DMEM, supplemented with 2 mg/ml bovine serum albumin, 2 mM L-carnitine, 5 mM creatine, 5 mM taurine, 0.1 µM insulin, and 10 nM triiodothyronine with 100 IU/ml penicillin and 100 µg/ml streptomycin. In experimental protocols designed to examine myocyte survival and/or apoptosis, insulin was omitted from the defined medium, which is therefore termed "ACCTT." Coronary microvascular endothelial cells (CMEC) from adult rat hearts were isolated as described by Nishida et al. (33). The isolated cells were plated at a density of 2500 cells/cm2. After 1 h of plating, the cells were washed twice with DMEM to remove loosely adherent cells and then maintained in DMEM supplemented with 20% FBS. These primary isolates have been documented to contain >90% endothelian cells, with a phenotype at low passage number consistent with their microvascular origin, as described previously (33).PCR Analysis of ErbB Receptors in Rat Heart-- cDNA sequences encoding portions of the C termini of ErbB receptors were amplified by using the following synthetic oligonucleotide primers: ErbB2A (5'-TGTGCTAGTCAAGAGTCCCAACCAC-3'; sense) and ErbB2B (5'-CCTTCTCTCGGTACTAAGTATTCAG-3'; antisense) for amplification of ErbB2 codon positions 857-1207 (34); ErbB3A (5'-GCTTAAAGTGCTTGGCTCGGGTGTC-3'; sense) and ErbB3B (5'-TCCTACACACTGACACTTTCTCTT-3'; antisense) for amplification of ErbB3 codon positions 712-1085 (35); ErbB4A (5'-AATTCACCCATCAGAGTGACGTTTGG-3'; sense) and ErbB4B (5'-TCCTGCAGGTAGTCTGGGTGCTG; antisense) for amplification of ErbB4 codon positions 896-1262 (36). RNA samples (1 µg) from rat hearts or freshly isolated neonatal and adult rat ventricular myocytes were reverse transcribed to generate first-strand cDNA. The PCR reactions were performed for 30 cycles. Each cycle included 30 s at 94 °C, 75 s at 63 °C, and 120 s at 72 °C. The PCR products were directly cloned into the TA cloning vector (Invitrogen Co., San Diego, CA) and verified by automatic DNA sequencing.
Analysis of ErbB Receptor Phosphorylation-- To analyze which receptor subtypes were tyrosine-phosphorylated, neonatal and adult ventricular myocyte cells were maintained in serum-free medium for 24-48 h and then treated with rhGGF2 at 20 ng/ml for 5 min at 37 °C. Cells were quickly rinsed twice with ice-cold PBS and lysed in cold lysis buffer containing 1% Nonidet P-40, 50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, 10 mM sodium molybdate, 8.8 g/liter sodium pyrophosphate, 4 g/liter NaFl, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 20 µM leupeptin. Lysates were centrifuged at 12,000 × g at 4 °C for 20 min, and aliquots of 1500 µg (ErbB4 detection) or 3000 µg (ErbB2 detection) of supernatant were incubated with antibody specific to ErbB4 or phosphotyrosine, respectively, overnight at 4 °C and precipitated with protein G plus agarose (Santa Cruz Biotechnology, Inc.; Santa Cruz, CA). Immunoprecipitates were collected and released by boiling in SDS sample buffer. Samples were fractionated by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes (Bio-Rad), and probed with a PY99 anti-phosphotyrosine antibody or with an antibody specific to ErbB2. All of these antibodies were purchased from Santa Cruz Biotechnology.
Incorporation of [3H]Thymidine and [3H]Leucine-- As an index of DNA synthesis, [3H]thymidine incorporation was measured as described previously (37). After incubation for 48-72 h in serum-free medium (DMEM plus ITS), the cells were stimulated with different concentrations of rhGGF2 (Cambridge NeuroScience Co., Cambridge, MA) for 20 h. [3H]Thymidine (0.7 Ci/mmol; DuPont) was then added to the medium at a concentration of 5 (NRVM) or 15 µCi/ml (embryonic myocytes), and the cells were cultured for another 8 h. Cells were washed with PBS twice and 10% trichloroacetic acid (Sigma) once, and 10% trichloroacetic acid was added to precipitate protein at 4 °C for 45 min. Parallel cultures of myocytes not exposed to rhGGF2 were harvested under the same conditions as controls. The precipitate was washed twice with 95% ethanol, resuspended in 0.15 N NaOH, and saturated with 1 M HCl, and then aliquots were counted in a scintillation counter. The results are expressed as relative cpm/dish normalized to the mean cpm of control cells in each experiment. For antibody-blocking experiments, the same procedure was applied except that the cells were preincubated with an antibody (0.5 µg/ml) specific for each neuregulin receptor (c-Neu Ab-2, Oncogene Research Products; and ErbB3 or ErbB4, Santa Cruz Biotechnology), for 2 h before the addition of either rhGGF2 or recombinant human fibroblast growth factor-2.
The rate of [3H]leucine uptake was used as an index of protein synthesis. For these experiments, 10 µM cytosine arabinoside was added to the culture medium. Cells were grown in serum-free medium for 36-48 h and then stimulated with different doses of rhGGF2. After 40 h, [3H]leucine (5 µCi/ml) was added for 8 h, and cells were washed with PBS and harvested with 10% trichloroacetic acid. Trichloroacetic acid-precipitable radioactivity was determined by scintillation counting as above.Indirect Immunofluorescence Staining of Cardiac Myocytes-- For examination of changes in myocyte phenotype with rhGGF2, cells were fixed in 4% (w/v) paraformaldehyde for 30 min at room temperature, rinsed with PBS, permeabilized with 0.1% Triton X-100 for 15 min, and then incubated with 1% FBS for another 15 min, followed by incubation with anti-MHC (1:300) and visualized with TRITC-conjugated (NRVM) or fluorescein isothiocyanate-conjugated (ARVM) second antibody. NRVM were examined using indirect immunofluorescence microscopy, while ARVM were examined using a MRC 600 confocal microscope (Bio-Rad) with a krypton/argon laser.
Cell Survival Assay and Detection of Apoptosis-- Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) cell respiration assay, which is dependent on mitochondrial activity in living cells (38). Primary cultures of NRVM after 2 days in serum-free medium were stimulated with different concentrations of rhGGF2 for either 4 or 6 days. ARVM were maintained in ACCTT medium or ACCTT medium plus different concentrations of rhGGF2 for 6 days. MTT was then incubated with the cells for 3 h at 37 °C. Living cells transform the tetrazolium ring into dark blue formazan crystals that can be quantified by reading the optical density at 570 nm after cell lysis with dimethyl sulfoxide (Sigma).
Apoptosis was detected in neonatal and adult myocytes using the terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) assay. 3'-End labeling of DNA with fluorescein-conjugated dUTP was done using an in situ cell death detection kit (Boehringer Mannheim) following the manufacturer's instructions. Cells were counterstained with an anti-MHC antibody as described above, and the nuclei were also stained with Hoescht 33258 (10 µM, Sigma) for 5 min. More than 500 myocytes were counted in each coverslip, and the percentage of TUNEL-positive myocytes was calculated. Flow cytometric analysis of neonatal myocytes fixed in 70% ethanol/PBS and stained with propidium iodide was also performed to quantify the percentage of cells undergoing apoptosis. This method is based upon the observation that cells undergoing apoptosis have a hypodiploid quantity of DNA and localize in a broad area below the G0/G1 peak on a DNA histogram. Briefly, cells were collected by trypsinization, pooled with nonattached cells, and fixed in 70% ethanol. After being rinsed once with PBS, cells were incubated with a propidium iodide (20 µg/ml, Sigma) solution containing RNase A (5 Kunitz units/ml) at room temperature for 30 min. Data were collected using a FACScan (Becton-Dickinson, San Jose, CA). For each sample, 10,000 events were collected. Aggregated cells and extremely small cellular debris were gated out.Isolation and Hybridization of RNA--
Total cellular RNA was
isolated by a modification of the acid guanidinium/thiocyanate
phenol/chloroform extraction method (39) using the TRIZOL reagent (Life
Technologies). The following cDNA probes labeled with
[-32P]dCTP by random priming were used: rat
prepro-atrial natriuretic factor (prepro-ANF) (0.6 kilobase pair of
coding region) (40), rat skeletal
-actin (240 base pairs of a
3'-untranslated region) (41), and rat heart neuregulin-1 (0.67 kilobase
pair of coding region spanning the Ig-like and epidermal growth
factor-like domains) (42). Rat glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) cDNA probe (240 base pairs of the coding region) (43) was
used as control for loading and transfer efficiency. Signal intensity was determined by densitometry (Ultrascan XL, Amersham Pharmacia Biotech).
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RESULTS |
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Expression of Neuregulin Receptors in the Heart-- To examine the expression profile of the NRG receptors (i.e. ErbB2, ErbB3, and ErbB4) in rat myocardium, RNA from rat heart tissues at midembryogenesis (embryonic day 14) or postnatal day 1 and from freshly isolated neonatal and adult ventricular myocytes were reverse transcribed and analyzed by RT-PCR. Primers that flank the variable C termini of ErbB receptors were used to amplify the products shown in Fig. 1A. In freshly isolated neonatal and adult rat ventricular myocyte primary cultures, both ErbB2 and ErbB4 mRNA were readily detectable by RT-PCR, although ErbB4 expression levels were consistently higher than those of ErbB2. Expression of ErbB3 mRNA was not detected in either whole heart at postnatal day 1 or freshly isolated cardiomyocytes, but ErbB3 mRNA was detected in the developing rat heart at embryonic day 14. Furthermore, when using receptor-specific cDNA probes for ErbB2, ErbB3, and ErbB4, only transcripts for ErbB4 were readily detectable in freshly isolated neonatal and adult rat ventricular myocytes but not in myocyte-depleted, non-myocyte-enriched primary cultures by Northern blot (data not shown).
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GGF2 Provokes Cardiac Myocyte Proliferation in Vitro-- To investigate the ability of GGF2 to stimulate the proliferative response of embryonic cardiac myocytes, myocytes maintained in serum-free medium for 3 days were subsequently treated with rhGGF2 for 30 h. As displayed in Fig. 2A, GGF2 provoked an approximately 2-fold increase in the DNA synthesis of rat embryonic cardiac myocytes (day 17), as assessed by [3H]thymidine incorporation. The maximal effective concentration was ~30 ng/ml (i.e. 0.54 nM). Exposure to recombinant human fibroblast growth factor-2 (25 ng/ml) had a similar effect. Since embryonic cardiac myocytes still possess active proliferative capacity, we next counted the cell number after a treatment with GGF2 (40 ng/ml) for the indicated time periods (Fig. 2B). The GGF-treated cells proliferated day by day, and the cell number peaked by day 4 and was increased by about 3-fold. However, as shown in Fig. 2C, GGF2 (50 ng/ml) had little mitogenic effect on cardiac non-myocyte fractions obtained following the preplating steps of the neonatal rat ventricular myocyte isolation procedure, while 7% FBS could induce a nearly 10-fold increase in [3H]thymidine incorporation into these cell populations. In neonatal ventricular myocytes, GGF2 could induce DNA synthesis to a lesser extent than in embryonic cardiac myocyte (Fig. 2, A and C). Therefore, GGF2 shows a relatively specific mitogenic effect on cardiac myocytes compared with a myocyte-depleted cell population, which, using the method of myocyte isolation we employed here, is composed largely of fibroblasts and endothelial cells (33).
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GGF2 Promotes Cardiac Myocyte Survival in Vitro-- During development, the net increase in the number of functional embryonic myocytes is dependent on both myocyte proliferative capacity and survival. Therefore, it was of interest to determine whether GGF2 could promote survival of cardiac myocytes in addition to proliferation. Primary cultures of neonatal rat ventricular myocytes maintained in serum-free media exhibited a gradual decrease in cell number. Using the MTT cell respiration assay to measure cell viability, we have observed that approximately 25% of cells die by day 4. In contrast, the addition of GGF2 resulted in a 30% increase in MTT activity compared with controls. The effect was concentration-dependent with an EC50 of 0.2 ng/ml (Fig. 3). This survival effect was observed in NRVM primary cultures for up to 7 days (data not shown); it was also observed in the presence of cytosine arabinoside, an antiproliferative agent. As shown in Fig. 3, the survival effect of GGF2 was observed at 4 days in the continuous presence of cytosine arabinoside, with about 90% myocyte viability in the presence of 50 ng/ml rhGGF2 compared with approximately 70% viability in control cultures. In contrast, GGF2 had no significant effect on the survival of myocyte-depleted, non-myocyte-enriched primary isolates at 4 days (data not shown).
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GGF2 Induces Hypertrophic Growth of Cardiac Myocytes--
To
investigate whether neuregulin signaling can induce a hypertrophic
(growth) response in cardiac myocytes, we examined the effects of GGF2
on induction of myocyte hypertrophy in both neonatal and adult rat
ventricular myocyte primary cultures. As shown in Fig.
6, A and B, after a
72-h incubation in serum-free medium with 20 ng/ml (i.e.
0.36 nM) of rhGGF2, NRVM exhibited a significant increase
in cell size and in myofibrillar development. A hypertrophic response
in cardiac myocytes is characterized by a number of phenotypic changes
in addition to an increase in cell size, such as an increase in
contractile protein content without cellular proliferation and the
reactivation of an "embryonic" gene program (44). Therefore, we
examined the effects of neuregulin on levels of prepro-ANF and skeletal
-actin mRNA (transcripts normally found in relatively low
abundance in neonatal and adult ventricular myocytes) and on
[3H]leucine incorporation as an index of protein
synthesis in NRVM primary cultures. As shown in Fig. 6C,
rhGGF2 (20 ng/ml) increased mRNA levels for prepro-ANF and skeletal
-actin within 60 min, approximately doubling by 16 h. GGF2 also
stimulated [3H]leucine incorporation, with about a 120%
increase at 48 h, at a concentration of 5 ng/ml. To minimize
possible confounding effects of GGF2 on the rate of
[3H]leucine uptake into non-myocyte contaminant cells,
these experiments were repeated in the continuous presence of cytosine
arabinoside with similar results (data not shown).
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CMEC Express NRG1: Regulation by Endothelin-- NRG1 expression has been identified in the developing murine (embryonic day 10 and 17) endocardial endothelium by in situ hybridization (11, 45). To determine if NRG1 expression could be detected in an endothelial cell phenotype obtained from the heart of adult animals, CMEC was isolated from adult rat ventricle and cultured for analysis of NRG1 expression. As shown in Fig. 8, a basal level of NRG1 mRNA was detected in confluent primary cultures of CMEC using a NRG1-specific cDNA probe that included both the Ig-like and epidermal growth factor-like domains. Consistent with a previous report (46), this probe recognizes three transcripts of 10, 7, and 3 kilobase pairs. In addition, as shown in Fig. 8 and in contrast to FBS, endothelin-1 increased NRG1 expression in primary CMEC cultures. We observed an induction of NRG1 mRNA (approximately 2-fold) that peaked at ~16 h and then decreased to basal levels by 24 h.
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DISCUSSION |
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The characterization of mice with targeted disruption of either the IgG-like or epidermal growth factor-like domains of the NRG1 gene and of mice expressing loss of function mutations of either the ErbB2 or ErbB4 gene, highlight the importance of neuregulins in cardiac development (11-14). In this report, we have documented that two neuregulin receptors, ErbB2 and ErbB4, continue to be expressed in the postnatal and adult myocardium. Further, we demonstrated the soluble neuregulin GGF2-induced phenotypic changes in neonatal as well as the "terminally differentiated" adult myocytes in culture.
The regulated expression of ErbB receptors presumably determines in part the developmental role of neuregulins during embryogenesis and in the postnatal heart. Using in situ hybridization, it had been reported that myocardial cells in the fetal heart expressed both ErbB2 and ErbB4 (11, 13) and that expression of ErbB3 was localized in the mesenchymal cells of the endocardial cushion and bulbus cordis (11) in midembryogenesis (embryonic day 10.5). The present study extends this observation to show that while all three receptors are expressed during midembryogenesis in the rat heart, only expression of ErbB3 is markedly down-regulated later during development. We also found that both ErbB2 and ErbB4 were rapidly and specifically phosphorylated in response to the addition of GGF2 to primary cultures of both neonatal and adult rat ventricular myocytes, suggesting ErbB2 and ErbB4 may form heterodimer to signal in postnatal myocardium.
In addition to their essential role during cardiac ontogeny,
neuregulins join a growing list of stimuli that induce hypertrophic growth of cardiac myocytes, including 1-adrenergic
agents, mechanical stretch, thyroid hormone, interleukin-1
,
fibroblast growth factors, heparin-binding epidermal growth factor-like
growth factor, endothelins, and angiotensins among others (2, 3, 6, 47,
48). What role GGF2 or other neuregulins play in late myocardial
embryogenesis or in the postnatal animal remains to be elucidated.
Since we observed not only myocyte growth but also reemergence of a
pattern of embryonic gene transcription in neonatal and adult
ventricular myocytes exposed to GGF2 that is characteristic of myocyte
hypertrophic growth in situ in the myocardium, it is
possible that neuregulins participate with other growth stimuli in some
forms of cardiac adaptation to physiologic stress in the postnatal
animal.
Unlike most other hypertrophic stimuli, however, GGF2 promoted the
survival of adult and neonatal cardiac myocytes, at least in part by
inhibiting apoptosis in low density, subconfluent myocyte cultures
maintained in serum-free medium. This result is similar to that
reported for CT-1, which, at least in neonatal rat ventricular myocytes, also has been shown to reduce apoptosis induced by the removal of serum (49). In contrast to the results reported for CT-1,
GGF2, at least under the conditions and at the concentrations we
employed, did not completely prevent apoptosis in neonatal myocytes,
although this may be due to methodological differences. The
antiapoptotic action of CT-1 has been shown to require the activation of the Ras/Raf/MEK/mitogen-activated protein kinase pathway,
since inhibition of extracellular signal regulated kinase 1 and 2 activation with the MEK inhibitor PD98059 prevented CT-1's antiapoptotic activity (49). Interestingly, GGF2 also activates these
mitogen-activated protein kinase pathways in
myocytes.2 However,
activation of this mitogen-activated protein kinase pathway alone
cannot be sufficient for the antiapoptotic effects of either of these
ligands, since this signal transduction cascade is also activated in
cultured cardiac myocytes by angiotensin II (5), as well as
interleukin-1 and IFN
(50), all of which have been shown to
promote apoptosis in both adult (51) and neonatal3 rat ventricular
myocytes.
This apparent paradox also is reflected in the effects of these
myocardial growth factors on the expression of ANF. Increased expression of ANF is one of the most reliable markers of hypertrophy of
ventricular myocytes, both in vivo and in vitro
(52). Interestingly, ANF has recently been reported to induce apoptosis
of neonatal rat ventricular myocytes when added to primary cultures at
sufficient concentrations (53). In contrast, while CT-1 and GGF2
increase the expression of ANF in neonatal myocyte cultures to levels
that are similar to those achieved with the hypertrophic but
pro-apoptotic stimuli, angiotensin II or interleukin-1/IFN
, both
CT-1 and GGF2 clearly protect against apoptotic myocyte death, at least in vitro.
Neuregulin-ErbB signaling may represent a generalized system for maintaining close apposition between distinct cell lineages that are necessary for normal tissue architecture and function in certain organs. Neuronally expressed neuregulins appear to drive the proliferation and growth and survival of adjacent Schwann cells during axonal generation (23, 26), and apoptosis of Schwann cells distal to mechanically injured motor axons can be prevented by application of a soluble neuregulins (25). Similarly, neuregulins expressed by motor neurons at the neuromuscular junction appear to drive the formation of the acetylcholine-responsive postsynaptic endplates in skeletal muscle (54). The expression of neuregulin by coronary microvascular endothelial cells suggest that these cells may play a role in the regulation of NRG synthesis and secretion in the postnatal myocardium that is analogous to that played by the endocardial endothelium in the heart during development. The effects of the soluble neuregulin, GGF2, on cardiac myocyte proliferation, growth, and survival in vitro offer additional insight into the role the neuregulin-ErbB signaling system plays in myocardial development. Persistent expression of neuregulin receptors in the adult heart and effects of this soluble neuregulin on the adult myocyte phenotype suggest that neuregulin signaling may be involved in cardiac adaptation to physiologic stress (i.e. ventricular "remodeling"). In addition, neuregulins appear to represent an additional component of an increasingly complicated cross-talk between endocardial/vascular endothelium and cardiac myocytes that remains poorly understood, both during development and in the postnatal animal.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant HL36141 (to R. A. K.).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.
These two authors contributed equally to this work.
§ Present address: Cardiology Division, Boston University Medical Center, 88 E. Newton St., Boston, MA 02118.
To whom correspondence should be addressed: Cardiology
Division, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115. Tel.: 617-732-7503; Fax: 617-732-5132; E-mail:
rakelly{at}bics.bwh.harvard.edu.
1 The abbreviations used are: NRG, neuregulin; ANF, atrial natriuretic factor; ARVM, adult rat ventricular myocyte(s); GGF, glial growth factor; rhGGF2, recombinant human glial growth factor 2; MHC, myosin heavy chain; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NRVM, neonatal rat ventricular myocyte(s); TRITC, tetramethylrhodamine isothiocyanate; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; CMEC, coronary microvascular endothelial cell(s); PBS, phosphate-buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR.
2 R. Baliga, D. Sawyer, Y.-Y. Zhao, R. Kelly, manuscript in preparation.
3 M. A. Arstall, D. B. Sawyer, and R. A. Kelly, submitted for publication.
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