(Circulation. 1999;100:407-412.)
© 1999 American Heart Association, Inc.
Basic Science Reports |
Neuregulin in Cardiac Hypertrophy in Rats With Aortic Stenosis
Differential Expression of erbB2 and erbB4 Receptors
From the Department of Medicine, Cardiovascular Division, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Mass (S.R., X.Y., E.O.W., F.H., J.B., B.H.L.); and Cambridge NeuroScience Inc, Cambridge Mass (M.A.M.).
Correspondence to Beverly H. Lorell, MD, Cardiovascular Division, Beth Israel Deaconess Medical Center, 330 Brookline Ave, Boston, MA 02215. E-mail blorell{at}caregroup.harvard.edu
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Abstract |
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Background—Neuregulins are a family of peptide growth factors that promote cell growth and viability. The potential role of neuregulin-erbB signaling in hypertrophic growth and later failure in the adult heart in vivo is not known.
Methods and Results—We used ribonuclease protection assays to quantify mRNA levels of neuregulin, erbB2, and erbB4 in left ventricular (LV) tissue and myocytes of normal rats and rats with aortic stenosis with pressure-overload hypertrophy 6 and 22 weeks after banding. At both stages of hypertrophy, Northern blot analyses of mRNA from LV myocytes showed upregulation of atrial natriuretic peptide, a molecular marker of hypertrophy (P<0.05). LV tissue neuregulin message levels were similar in animals with aortic stenosis compared with controls (P=NS) and were not detectable in myocytes. LV erbB2 and erbB4 message levels in LV tissue and myocytes were maintained during early compensatory hypertrophy in 6-week aortic stenosis animals compared with age-matched controls; in contrast, erbB2 and erbB4 message levels were depressed in 22-week aortic stenosis animals at the stage of early failure (both P<0.01 vs age-matched controls). Immunoblotting of erbB2 and erbB4 also showed normal protein levels in 6-week aortic stenosis animals compared with controls; however, erbB2 and erbB4 protein levels were depressed in 22-week aortic stenosis animals (48% decrease in erbB2, P<0.05, and 43% decrease in erbB4, P<0.01) relative to age-matched controls.
Conclusions—The neuregulin receptors erbB2 and erbB4 are downregulated at both the message and protein levels at the stage of early failure in animals with chronic hypertrophy secondary to aortic stenosis. These data suggest a role for disabled erbB receptor signaling in the transition from compensatory hypertrophy to failure.
Key Words: growth substances • myocytes • hypertrophy • heart failure
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Introduction |
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Locally acting polypeptide growth factors, synthesized by the cardiac myocyte itself or adjacent endothelial cells, are differentially expressed during cardiac development and pathological pressure-overload hypertrophy.1 2 3 4 5 Neuregulins (also called NRGs or glial growth factors)6 7 are a large group of membrane-bound or secreted peptides that promote growth, differentiation, and survival during development and oncogenesis.8 9 10 11 Although 3 neuregulin genes have been identified, the NRG-1 gene and its products have been studied most extensively.12 13 14 The neuregulins are agonists for the epidermal growth factor (EGF) family of tyrosine kinase receptors, which include the EGF receptor (EGFR), erbB2, erbB3, and erbB4 receptors. Ligand binding promotes combinatorial receptor interactions, including the formation of erbB4 homodimers and heterodimers involving other receptor subunits.13
Biological functions of neuregulin-erbB signaling have been studied through in vitro and in vivo pharmacological experiments with the use of recombinant neuregulins, by the characterization of mice with targeted gene disruptions, and from analyzing expression patterns of the ligands and the receptors in embryonic development and in animal models of disease and injury (see References 6, 9, and 156 9 15 for review). These studies have provided numerous examples of bioactivities signaled through the neuregulin-erbB system, such as cell survival, proliferation, migration, and differentiation, which are essential for normal development and differentiated function in a variety of tissues, including the heart. For example, neuregulin signaling has been shown to suppress apoptosis and promote cell survival in various glial cells such as astrocytes,16 oligodendrocytes,17 retinal ganglion cells,18 and Schwann cells19 20 21 and in cultured cardiomyocytes.22 Furthermore, Zhao et al22 reported that erbB2 and erbB4 receptors are expressed in both neonatal and adult rat myocytes in culture and showed that neuregulin-erbB receptor signaling in these in vitro systems suppresses apoptosis and promotes myocyte growth and survival. We hypothesized that the neuregulin-erbB receptor system may be implicated in pathological hypertrophy in vivo in the adult heart. Therefore we examined left ventricular (LV) message levels of neuregulin (NRG-1) and the erbB2 and erbB4 receptor genes and proteins in aortic stenosis rats at the stages of early pressure-overload hypertrophy and later failure in comparison with age-matched controls.
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Methods |
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Aortic Stenosis Model
Ascending aortic stenosis was performed in male Wistar weanling rats (body weight 50 to 70 g, age 3 to 4 weeks, obtained from Charles River Breeding Laboratories, Wilmington, Mass), as previously described by our laboratory.4 5 23 24 25 26 Sham-operated animals served as age-matched controls. Aortic stenosis animals and age-matched sham-operated controls were killed after administration of anesthesia with intraperitoneal pentobarbital 65 mg/kg at 6 and 22 weeks after surgery (n=20 to 29 per group). Our hemodynamic and echocardiographic studies in this model have shown that compensatory hypertrophy with normal LV cavity dimensions and contractile indexes is present 6 weeks after banding, whereas animals develop early failure by 22 weeks after banding, which is characterized by onset of LV cavity enlargement and mild depression of ejection indexes and pressure development per gram of LV mass.4 5 23 24 25 26 In the present study, in vivo LV pressure measurements were performed before the animals were killed, as previously described.4 5 23 24 25 26 The animals were also inspected for clinical markers of heart failure, including the presence of tachypnea, ascites, and pleural effusions. Both body weight and LV weight were recorded.
LV Myocyte Isolation for RNA Extraction
In a subset of animals (n=10 per group), the heart was rapidly
excised and attached to an aortic cannula. Myocyte dissociation by
collagenase perfusion was performed as previously described
by our laboratory.27 28 29 To evaluate the percentage of
myocytes in the final cell suspension, aliquots of myocytes were fixed,
permeabilized, and blocked. The cell suspension was
then incubated with antibodies against 
-sarcomeric actin (mAb,
Sigma, 1:20) and von Willebrand factor (pAb, Sigma, 1:200) to
distinguish between myocytes and endothelial cells.
Secondary antibodies (goat anti-rabbit, goat anti-mouse pAb, molecular
probes, 1:400) with a Texas red (or Oregon green) conjugate were used
as a detection system. We routinely obtained 98% myocytes and <2%
fragments of endothelial cells or unstained cells
(fibroblasts), respectively.
Ribonuclease Protection Assay
Total RNA was isolated from control and hypertrophied myocytes
(n=10 hearts in each group) and from LV tissue (n=10 hearts in each
group) with the use of TRI Reagent (Sigma). Tissue and myocyte RNA were
used for the following protocols. With the use of myocyte RNA, Northern
blots were used to assess message levels of atrial
natriuretic peptide that were normalized to
GAPDH.23 29 These experiments were done to confirm the
specificity of myocyte origin of the RNA with the use of this molecular
marker of hypertrophy.
We also performed reverse transcription–polymerase chain reaction
(RT-PCR) for initial estimation of the presence of erbB2, erbB4, and
neuregulin in samples derived from adult rat heart and adult myocytes
by using the following pairs of primers: erbB2 sense 5' GCT GGC TCC GAT
GTA TTT GAT GGT 3', erbB2 antisense 5' GTT CTC TGC CGT AGG TGT CCC
TTT 3',30 erbB3 sense 5' GCT TAA AGT GCT TGG CTC GGG TGT C
3', erbB3 antisense 5' TCC TAC ACA CTG ACA CTT TCT CTT
3',31 erbB4 sense 5' AAT TCA CCC ATC AGA GTG ACG TTT GG
3', erbB4 antisense 5' TCC TGC AGG TAG TCT GGG TGC TG
3',32 neuregulin sense 5' GCA TCA CTG GCT GAT TCT
GGA G 3', neuregulin antisense 5'CAC ATG CCG GTT ATG GTC AGC A
3'. The latter primers recognize the NRG-1 gene but do not discriminate
between its isoforms. The amplification was initiated by 1 minute of
denaturation, 2 minutes of annealing at the gene-specific temperature,
and 2 minutes of extension at 72°C. The whole PCR reaction was
electrophoresed on a 1% agarose gel and the PCR products of
expected size were gel-purified. After cloning these fragments into
pGEM-T vector (Promega), the correctness and orientation of those
fragments within the vector were confirmed by sequencing. Cloned PCR
fragments were used to generate a radiolabeled riboprobe with the use
of the MAXIscript in vitro transcription kit (Ambion) and
-32P-UTP. The plasmids containing the erbB2,
erbB4, or neuregulin fragment were linearized and a radiolabeled probe
was synthesized by in vitro transcription with T7 or T3 RNA polymerase.
The ß-actin probe provided by the kit was transcribed with T7 or T3
polymerase and resulted in a 330 and 300-bp fragment, respectively.
Twenty micrograms of total RNA was hybridized to
5x105 cpm of erbB2, erbB4, or neuregulin c-RNA
together with 2x104 cpm of ß-actin for later
normalization according to the RPA II kit (Ambion) protocol.
After digestion with RNase A/RNase T1, the samples were precipitated,
dried, redissolved, and finally separated on a 5%
polyacrylamide gel for 2 hours. The gel was exposed to Kodak MR
film for 12 to 48 hours, and the assay was quantified by densitometric
scanning of the autoradiograph with Image Quant software (Molecular
Dynamics). erbB2, erbB4, and neuregulin mRNA levels were normalized to
ß-actin.
Western Blotting of erbB2 and erbB4
LV tissue (n=5 hearts per group) was rapidly
homogenized in a RIPA buffer containing 50 mmol/L Tris
HCl, pH 7.4, 1% NP-40, 0.1% SDS, 0.25% Na-deoxycholate, 150
mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L PMSF, 1 µg/mL
aprotinin, 1 µg/mL leupeptin, 1 µg/mL pepstatin, and 1 mmol/L
Na3PO4. Proteins were
quantified with the Lowry assay kit (Sigma). Fifty micrograms of
protein in Laemmli SDS sample buffer was boiled for 5 minutes and after
centrifugation loaded onto a 10% SDS-PAGE gel. After
electrophoresis, proteins were transferred to a nitrocellulose membrane
at 100 mA overnight. The filters were blocked with 0.05% Tween-20, 5%
nonfat milk and then incubated with anti-erbB2 or anti-erbB4 (Santa
Cruz Biotechnology, each diluted 1:100, 1 µg/mL). After incubation
with goat anti-rabbit peroxidase-conjugated secondary antibody diluted
1:2000, blots were subjected to the enhanced chemiluminescent detection
method (Amersham, Life Science) and afterward exposed to Kodak MR film
for 30 to 180 seconds. Protein levels were normalized to protein levels
of ß-actin detected with anti-ß-actin (Sigma).
In Situ Hybridization for Neuregulin
Ten-micron cryostat sections of LV tissue (n=2 control and
6-week aortic stenosis hearts) were used for in situ
hybridizations. Antisense and sense RNA probe was synthesized from cDNA
fragments in pBluescript with either T7 or T3 RNA polymerase and
digoxigenin-labeled UTP (DIG RNA Labeling Mix, Boehringer
Mannheim). Tissue sections were first treated with 4%
paraformaldehyde for 20 minutes, followed by 30 minutes
digestion with proteinase K (10 µg/mL) at 37°C and another 5
minutes of fixation in 4% paraformaldehyde. After
fixation, the slides were washed in PBS 3 times for 5 minutes.
Afterward the sections were immersed in 0.1 mol/L triethanolamine
chloride buffer with 0.25% acetic anhydride for 10 minutes to block
polar and charged groups on the section and hence to prevent
nonspecific probe binding. The in 2x SSC washed slides were then
prehybridized (50% formamide, 2x SSC, 5% dextran sulfate, 0.1% SDS,
1x Denhardt's, 400 µg/mL denatured salmon sperm DNA) at 45°C for
60 minutes in a moist chamber charged with 50% formamide/2x SSC.
After 1 hour the probes were added to the solution and the slides were
hybridized for 16 to 18 hours at 45°C. After overnight hybridization,
slides were washed twice in 4x SSC for 30 minutes at 45°C under
shaking and then incubated with RNAseA (40 µg/mL) in 500 mmol/L
NaCl, 10 mmol/L Tris, 1 mmol/L EDTA, pH 8.0, for 30 minutes
at 37°C to remove unhybridized probe. After RNase treatment, sections
were immersed in 2x SSC at 50°C for 30 minutes and in 0.2x SSC at
the same temperature for another 30 minutes. The slides were
equilibrated with TBS I buffer (100 mmol/L Tris, 150 mmol/L
NaCl, pH 7.5) and then blocked with blocking reagent for 30 minutes at
room temperature according to the manufacturer's protocol (DIG Nucleic
Acid Detection Kit, Boehringer Mannheim). After the blocking
reagent was removed the slides were immersed in TBS I for 1 minute and
then the anti–DIG-AP conjugate solution (DIG Nucleic Acid Detection
Kit, Boehringer Mannheim) was applied to each section for 1.5
hours at room temperature in a humid chamber. Afterward the slides were
washed in TBS I 3 times for 10 minutes to wash off the excess of
antibody and equilibrated in TBS II (100 mmol/L Tris, 100
mmol/L NaCl, pH 9.5, 50 mmol/L MgCl2 0.7
H2O) for 5 minutes. The color substrate was
prepared according to the manufacturer's instructions and applied to
each section until a blue-colored reaction became visible. The reaction
was stopped and the slides were washed in PBS and distilled water for 5
minutes each. After a nuclear counterstaining the sections were
dehydrated through an ethanol series, immersed in xylene, and mounted
by cover slipping in Permount (Fisher Scientific).
Statistical Analysis
All values are expressed as mean±SEM. Statistical
analysis of differences observed between the aortic
stenosis groups (6 and 22 weeks after banding) and the
age-matched control groups was done by ANOVA comparison. An unpaired
Student's test was used for comparison among the groups at the same
age after banding. Statistical significance was accepted at the level
of P<0.05.
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Results |
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LV Hypertrophy and Hemodynamics
As shown in Table 1
, LV
weight and the LV/body weight ratio were significantly
(P<0.05) increased in the 6-week and 22-week aortic
stenosis animals compared with age-matched controls. The in
vivo LV systolic pressure was significantly increased in both
6-week and 22-week aortic stenosis animals compared with
age-matched controls. In vivo LV end-diastolic pressure was
also higher in aortic stenosis animals compared with
age-matched controls. Consistent with prior studies in this
model, LV systolic developed pressure per gram was
significantly higher in 6-week aortic stenosis animals in
comparison with age-matched controls but depressed in 22-week aortic
stenosis animals. At 22 weeks after banding, the aortic
stenosis animals also showed clinical markers of failure
including tachypnea and small pleural and pericardial effusions.
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Expression of LV erbB2, erbB4, and Neuregulin in Aortic
Stenosis
We were able to detect erbB2, erbB4, and neuregulin mRNA but
not erbB3 message in LV tissue derived from hearts of adult male rats
with and without LV hypertrophy as well as in RNA from
normal and hypertrophied myocytes by RT-PCR. Steady-state levels of
erbB2, erbB4, and neuregulin mRNA levels in LV tissue from aortic
stenosis rats and controls (n=5 hearts per group) were then
measured by ribonuclease protection assay (RPA) and normalized to
ß-actin. The LV neuregulin mRNA levels were not significantly
different in tissue from 6-week compared with age-matched controls
(0.68±0.12 vs 0.45±0.12 U, NS) or 22-week aortic stenosis
rats compared with age-matched controls (0.78±0.21 vs 0.51±0.21 U,
NS). The LV erbB2 and erbB4 mRNA levels, which were normalized to
levels of ß-actin, were preserved in 6-week aortic stenosis
rats with compensatory hypertrophy relative to controls.
However, LV erbB2 (P<0.05) and erbB4 (P<0.01)
message levels were significantly depressed in 22-week aortic
stenosis rats at the stage of early failure (Figure 1 and Table 2).
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We next examined gene expression in RNA from LV myocytes of 6-week and
22-week aortic stenosis animals and controls. The specificity
of expression in myocytes was determined by examining message levels of
atrial natriuretic peptide (ANP), a positive molecular
marker of pressure-overload hypertrophy, with the use of
myocyte RNA and normalization to levels of GAPDH. As shown in Figure 2, ANP was upregulated in myocytes from
both 6-week (710±16 vs 230±40 U, P<0.05) and 22-week
aortic stenosis animals (898±52 vs 339±13 U,
P<0.05) in comparison with controls (n=5 per group).
Neuregulin was not detectable by RPA in RNA derived from myocytes in
any group. erbB2 (n=5 per group) and erbB4 (n=3 to 4 per group) message
levels were also measured in myocyte RNA from both aortic
stenosis groups (Figure 3 and
Table 2
). Consistent with the measurements in LV tissue
samples, erbB2 myocyte message levels were preserved in 6-week aortic
stenosis animals relative to age-matched controls (NS) and
depressed in 22-week aortic stenosis relative to age-matched
controls (P<0.01). erbB4 myocyte message levels were
preserved in 6-week aortic stenosis animals relative to
age-matched controls (NS) and depressed in 22-week aortic
stenosis relative to age-matched controls
(P<0.01).
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LV erbB2 and erbB4 Protein Levels
Western blotting with the use of polyclonal antibodies for erbB2
and erbB4 was performed with protein samples derived from LV tissue of
6-week and 22-week aortic stenosis rats in comparison with
age-matched controls (n=5 per group). Densitometric signals were
normalized relative to levels of ß-actin protein. As shown in Figure 4 and Table 2, erbB2 and
erbB4 LV protein levels were preserved in 6-week aortic
stenosis animals compared with age-matched controls. However,
both erbB2 and erbB4 LV protein levels were severely depressed in
22-week aortic stenosis animals relative to controls. Thus a
decrease in both LV message and protein levels of erbB2 and erbB4 is
present at the stage of early failure in this model of pressure
overload.
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In Situ Hybridization for Neuregulin
Antisense digoxigenin-labeled mRNA of neuregulin generated
reproducible hybridization signals on LV cryosections, whereas the
corresponding sense transcript generated no signal above background.
Neuregulin signals in adult heart cryosections were observed in the
endothelial cells of the cardiac microvasculature with
minimal or no signal in other cell compartments (micrographs not
shown). There was no difference between control and aortic
stenosis animals.
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erbB Signaling and Myocardial Viability
The neuregulins and their receptors are implicated in the regulation of cell survival and proliferation both in cancer cells and normal neural cells. Several types of solid tumors express erbB receptors, and a monoclonal antibody directed against the erbB2 receptor (anti-HER2 antibody) promotes tumor cytotoxicity in patients with breast cancer.33 34 Interestingly, O'Rourke et al35 recently studied human glial cells, which are highly resistant to radiation injury, and showed that disabling erbB receptors markedly sensitized cells to injury and apoptosis in response to stress, including radiation. Conversely, neuregulin-erbB signaling promotes cell survival during nervous system development and confers cell protection and function to nerves damaged by injury,19 20 21 neurotoxic compounds, or autoimmunity against myelin.20 36 Recent studies suggest that neuregulin-erbB signaling is also important during cardiac development and may afford cardioprotection in response to injury. Transgenic experiments in mice demonstrate that neuregulin-erbB receptor signaling is required for normal cardiac trabeculation and morphogenesis.37 38 39 40 Zhou et al22 have shown that erbB2 and erbB4 receptors are expressed in neonatal and adult rat myocytes and that neuregulin-erbB signaling promotes cell viability and protects against apoptosis in short-term studies in cultured cells. Congruent with the studies in nerve injury models, these in vitro cultured myocyte data support the hypothesis that neuregulin-erbB receptor signaling protects against myocyte injury and promotes viability.
In the present study, we used the well-characterized aortic stenosis rat model of LV pressure-overload hypertrophy and demonstrated that the neuregulin-erbB pathway may be implicated in vivo in hypertrophy and transition to early failure. This study showed that the neuregulin receptors erbB4 and erbB2 are normally expressed at the stage of early compensatory hypertrophy in 6-week aortic stenosis animals compared with age-matched controls. In contrast, in 22-week aortic stenosis animals at the stage of early failure, both LV message and protein levels of erbB4 and erbB2 are severely depressed in comparison with age-matched controls. It appears that the cardiac expression of erbB4 and erbB2 is not related to the extent of hypertrophy per se, since the magnitude of hypertrophy as well as expression of the molecular marker of hypertrophy, atrial natriuretic peptide, were similar at both stages of aortic stenosis.
Our study also demonstrated that neuregulin message was detected by RPA in LV tissue from normal and hypertrophied animals. It was not present in sufficient abundance to be detected by RPA in RNA from isolated myocytes. In corroboration, in situ hybridization studies indicated that neuregulin is predominantly localized in the endothelial compartment of the microvasculature of the adult rat heart. Developmental studies demonstrate that neuregulin is expressed in endothelial-endocardial cells and cushion mesenchyme, although the expression pattern of neuregulin isoforms during cardiac development and in the adult heart is not yet known.37 Thus similar to other growth-signaling systems such as endothelin-1 and angiotensin II,4 it is likely that neuregulin is predominantly expressed by endothelial and endocardial cells and modifies growth of adjacent target myocardial cells by a paracrine or juxtacrine mechanism.
Taken together, our findings and prior work in in vitro models raise the hypothesis that disabled erbB signaling results in a loss of cardioprotection in the hypertrophied heart and contributes to development of early failure. In chronic hypertrophy, both mechanical stress and secondary expression of proteins such as tumor necrosis factor and angiotensin II may damage myocardial cells and increase the risk of apoptosis.40 41 42 Recent startling clinical observations support the speculation that disabling cardiac neuregulin-erbB signaling greatly increases the susceptibility of the heart to injury and subsequent development of heart failure. In clinical trials of patients with breast cancer treated with doxorubicin plus Herceptin, a monoclonal antibody directed against the erbB2 receptor, only 3% of patients treated with doxorubicin alone developed severe class III or IV heart failure compared with 19% of patients treated with anti-erbB2 monoclonal antibody and doxorubicin.43 A limitation of the present study is that our findings do not demonstrate a critical cardioprotective role of the neuregulin-erbB signaling pathway in vivo regarding myocardial viability in this model of hypertrophy. Future studies with conditional cardiocyte-targeted overexpression and knockout of erbB2 and erbB4 receptors in transgenic adult mice will be required to rigorously prove or refute this hypothesis.
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Acknowledgments |
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This study was supported in part by NHLBI grant HL-38189 (Drs Lorell and Weinberg), by a NASA Award (Dr Lorell), by Fellowship Award F05 TWO05261-01 from the Fogarty International Center, the NIH (Dr Bartunek), and by a Konrad Adenauer Fellowship (S. Rohrbach). An educational grant for development of the aortic stenosis animal colonies used in this study was provided by Cambridge Neuroscience, Cambridge, Mass. We appreciate the assistance of Lois Wiltberger in preparation of the manuscript. We also appreciate the helpful suggestions of Ralph Kelly, MD, in discussion of these experiments.
Received July 31, 1998; revision received March 30, 1999; accepted April 9, 1999.
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