(Circulation. 1999;99:441-447.)
© 1999 American Heart Association, Inc.
Basic Science Reports |
RGS4 Inhibits G-Protein Signaling in Cardiomyocytes
From the Center for Cardiovascular Research, Department of Medicine, and the Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, Mo.
Correspondence to Anthony J. Muslin, Center for Cardiovascular Research, Box 8086, Washington University School of Medicine, 660 S Euclid Ave, St Louis, MO 63110. E-mail amuslin{at}im.wustl.edu
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Abstract |
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Background—RGS family members are GTPase-activating proteins for heterotrimeric Gq and Gi proteins. RGS genes are expressed in heart tissue and in cultured cardiomyocytes. There is evidence that altered RGS gene expression may contribute to the pathogenesis of cardiac hypertrophy and failure.
Methods and Results—We investigated the ability of RGS proteins to block G-protein signaling in vivo by using a cultured cardiomyocyte transfection system. Endothelin-1, angiotensin II, and phenylephrine signal through Gq or Gi family members and promote the hypertrophy of cardiomyocytes. We found that phenylephrine-mediated and endothelin-1–mediated induction of the atrial natriuretic factor and myosin light chain-2 genes was inhibited in cells that were transfected with RGS4. Phenylephrine-mediated gene induction was not inhibited in cells that were transfected with N128A-RGS4, a point mutant form that lacks GTPase-activating protein activity. Phenylephrine-mediated myofilament organization and cell growth were also blocked in cells by RGS4.
Conclusions—These results demonstrate that RGS protein can inhibit G-protein–mediated signaling in vivo and suggest that increased expression of RGS protein may be a counterregulatory mechanism to inhibit G protein signaling.
Key Words: hypertrophy • genes • growth substances • atrial natriuretic factor • proteins
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Introduction |
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Postnatal mammalian cardiomyocytes respond to a variety of stimuli, including mechanical stress, growth factor and hormonal action, and metabolic abnormalities, by increasing their cell size.1 2 3 Postnatal cardiomyocytes have the capacity to hypertrophy but are unable to proliferate for reasons that are not understood. Cardiomyocyte hypertrophy leads to growth of the entire heart, resulting in thickening of the ventricular chambers. Cardiac hypertrophy is an independent risk factor for the development of serious cardiac arrhythmias4 5 and is associated with diastolic dysfunction that can result in congestive heart failure.6 7
In cultured cardiomyocytes, mechanical stress and ligands such as phenylephrine,8 9 endothelin-1,10 angiotensin II,11 and basic fibroblast growth factor (bFGF)12 promote a hypertrophic response. This response is characterized by an increase in cell size, protein synthesis, and organization of contractile proteins into sarcomeres and by an induction of specific genes.13 Genes that are induced include atrial natriuretic factor (ANF),14 the immediate early proto-oncogene c-fos,15 and myosin light chain-2 (MLC-2).16 There is evidence that the action of these ligands on cultured cells mimics cardiac hypertrophy in whole animals. For example, in aortic-banded rats, treatment with an angiotensin-converting enzyme inhibitor at a dosage that does not reduce blood pressure causes regression of cardiac hypertrophy.17 Treatment of banded rats with the vasodilator hydralazine does not result in regression of hypertrophy.
Three agonists that cause cultured cardiomyocytes to
hypertrophy signal through heterotrimeric G proteins.
Endothelin-118 and angiotensin
II19 bind to 7 transmembrane receptors that are coupled to
Gq proteins, whereas phenylephrine
binds to 
1-adrenergic receptors that are
coupled to Gi and Gq
proteins.20 It is interesting to note that mechanical
stress may lead to the local release of angiotensin II or
endothelin-1 in the heart.21 FGF does not signal through
heterotrimeric G proteins but instead activates a signaling
cascade that includes the proteins FRS2, Grb2, SOS, and the small G
protein ras.22
A family of mammalian signaling molecules was recently identified
and termed RGS (for regulators of G-protein signaling), based on
homology to the budding yeast protein Sst2.23 Genetic
studies in yeast revealed that Sst2 negatively regulates signaling by
heterotrimeric G proteins. Biochemical studies performed in vitro with
purified proteins have demonstrated that RGS proteins have
GTPase-activating protein (GAP) activity toward -subunits of
heterotrimeric G proteins of the Gi and
Gq families.24 25 26 27 RGS proteins
promote the rapid deactivation of G proteins by binding to and
stabilizing the transition state of -subunits as they hydrolyze GTP.
The biological role of RGS proteins in mammalian tissues is largely
unknown.
We have recently demonstrated that the RGS3 and RGS4 genes are expressed in heart.28 We also found that RGS gene expression is enhanced in hypertrophied cultured cardiomyocytes and in cardiac tissues from pulmonary artery–banded mice.28 These experiments suggest that RGS function may be regulated at the transcriptional level; however, they do not establish whether increased RGS expression promotes, inhibits, or has no effect on the hypertrophic growth program.
We hypothesized that RGS family members are intrinsic cardiac proteins that block hypertrophy by inhibiting signal transduction stimulated by ligands such as phenylephrine and endothelin-1. To test this hypothesis, we overexpressed RGS4 in neonatal rat cardiomyocytes that were subsequently stimulated with hypertrophic ligand. Our results demonstrate that RGS4 can inhibit the action of phenylephrine and endothelin-1 but not bFGF in cultured cardiomyocytes.
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Methods |
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Reagents
Collagenase was obtained from Wako Pure Chemicals, Inc. Dulbecco's modified Eagle medium and other tissue culture material was from Life Technologies, Inc. The murine monoclonal anti–myc-1-epitope antibody was obtained from Babco. The rabbit polyclonal anti-RGS4 antibody was generated as previously described.28 Fluorescein isothiocyanate (FITC)-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology. Phenylephrine, endothelin-1, FGF, human apotransferrin, insulin, bovine serum albumin, and phorbol 12-myristate 13-acetate (TPA) were obtained from Sigma. Tetramethylrhodamine isothiocyanate (TRITC)-labeled phalloidin was obtained from Molecular Probes, Inc. ß-Galactosidase assays were performed with the Galactolight Kit from Tropix, and luciferase assays were performed with a kit from Analytical Luminescence Laboratory.
Expression and Reporter Plasmids
The open reading frame of rat RGS4 or a point mutant form
of RGS4 (N128A) linked in-frame to the myc-1 epitope was
subcloned into the pCR3 vector that contains a strong cytomegalovirus
(CMV) promoter and is designed for high-level expression in mammalian
cells. These vectors have been named pCMV-RGS4-myc and
pCMV-N128A-RGS4-myc. The luciferase reporter construct
pANF(-638)L
5' was kindly provided by Dr K.R. Chien (Department of
Medicine, University of California, San Diego).14 The
luciferase reporter construct pMLC(-250)L5' was a gift from Dr A.
Thorburn (University of Utah, Salt Lake City).13 To
control for cardiomyocyte transfection efficiency, pON249,
a ß-galactosidase expression vector under control of the human
cytomegalovirus promoter, was used as previously
described.14
Myocardial Cell Cultures
Cultured rat neonatal cardiomyocytes were prepared
as described.29 Briefly, ventricles were obtained from 1-
to 2-day-old Sprague-Dawley pups, and cardiomyocytes were
isolated by digestion with collagenase (Wako).
Cardiomyocytes were separated from nonmyocytes by differential
plating. The estimated purity of cardiomyocytes after
differential plating was 90% to 95%. Purified
cardiomyocytes were plated on collagen-coated dishes
at low density29 (200 cells/mm2) in
primary myocyte medium containing Dulbecco's modified Eagle medium
supplemented with 10% donor horse serum, 5% fetal calf serum,
glutamine, 0.1 mmol/L 5-bromo-2'-deoxyuridine (BrdU) and
antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin) and
maintained at 37°C in a humidified atmosphere of 5% carbon dioxide.
Twelve hours later, the cells were washed twice with Hanks' balanced
salt solution (without calcium, magnesium, or phenol red) and
resuspended in serum-containing medium. Twenty-four hours after
plating, the cells were washed and resuspended in serum-free medium
(Dulbecco's modified Eagle medium supplemented with insulin,
apotransferrin, bovine serum albumin, BrdU, and antibiotics as
above). To induce hypertrophy, phenylephrine
(100 µmol/L), endothelin-1 (10 nmol/L), or bFGF (25 ng/mL) was
added to the maintenance medium and cells were incubated for
40 hours.
Transient Transfection of Cultured Neonatal Rat
Ventricular Myocytes
Ventricular myocytes were plated as described above.
Immediately after plating, transient transfection was carried out with
the synthetic liposomal agent DOTAP
(N-[1-(2,3-dioleoyloxy)propyl]-N, N,
N-trimethylammonium methyl sulfate) (Boehringer
Mannheim) according to the manufacturer's instructions. Briefly,
plasmid DNA in 20 mmol/L HEPES was incubated for 15 minutes at
room temperature with DOTAP. The reaction mixture was then slowly added
to the primary myocyte medium and incubated overnight. For each
transfection, 1.5 µg of reporter plasmid, 1.5 µg of
ß-galactosidase plasmid, and/or 0 to 15 µg of
pCMV-RGS4-myc plasmid was used.
Luciferase and ß-Galactosidase Assays
After agonist stimulation, cells were washed twice with PBS
(without added calcium or magnesium) and were lysed with 400 µL of
reporter lysis buffer per 3.8 cm2 well (Promega).
Cell lysates were subjected to 1 round of freeze-thawing. Supernatants
were used in ß-galactosidase assays (Tropix) and luciferase assays
(Analytical Luminescence Laboratory) according to the manufacturer's
instructions. All activities were measured in duplicate with a
monolight 401 luminometer (Analytical Luminescence Laboratory).
Luciferase activity was normalized by ß-galactosidase activity for
each sample in all experiments to correct for any variation in
cardiomyocyte transfection efficiency.13 14
Results are expressed as the percent of maximal induction in control
versus agonist-treated cells. Results are presented as mean±SE
for experiments on 3 separate preparations of
cardiomyocytes.
Immunofluorescence Microscopy
Cardiomyocytes were cultured on collagen-precoated, plastic,
2-well chamber slides (Labtek). Cells were washed twice with PBS and
fixed in 3% paraformaldehyde. After fixation, cells
were permeabilized with 1% Igepal CA-630 (Sigma) in
PBS for 5 minutes at room temperature and nonspecific binding sites
were blocked with 10% horse serum in 1% Igepal CA-630 in PBS for 10
minutes at room temperature. Primary antibodies were diluted 1:200 in
PBS-glycine (10 mmol/L) and incubated at room temperature for 1
hour. Secondary FITC-conjugated or TRITC-conjugated antibodies (1:200
dilution) were used for immunofluorescence and
incubated for 30 minutes at room temperature. TRITC-labeled phalloidin
was diluted 1:50 in PBS-glycine before use. Cells were washed 3 times
with PBS-glycine (10 mmol/L) after each antibody incubation step
and were visualized by immunofluorescence
microscopy.
Analysis of RGS4-myc Expression
Levels
To determine the level of RGS4-myc expression
achieved by transfection with plasmid DNA, cardiomyocytes
were transfected with 1.5 µg of ß-galactosidase plasmid and 1.5
µg of pCMV-RGS4-myc and were incubated for 40 hours.
Double-immunofluorescence microscopy was performed
by use of anti–ß-galactosidase primary antibody to identify
transfected cells and by use of anti-RGS4 polyclonal primary antibody
to determine RGS4 protein levels in transfected versus nontransfected
cells. Digitized immunofluorescent photomicrographs of cells
were downloaded onto a Power MacIntosh computer and analyzed
with NIH Image software. The mean signal intensity of 10 to 15
transfected to nontransfected cells was compared in 2 separate
experiments.
Statistical Analysis
Analyses between groups were performed with the use of
ANOVA, with the post hoc Scheffé's test, with a value of
P<0.05 considered significantly different.
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Results |
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Subcellular Localization of Native and Transfected RGS4 Proteins in Neonatal Cardiomyocytes
Previous work has demonstrated that heterotrimeric G-protein subunits are primarily localized to the plasma membrane and the cytoplasmic compartments of cultured cells.30 To determine the subcellular localization of native RGS4 protein, untransfected cardiomyocytes were probed with purified polyclonal anti-RGS4 antibody followed by secondary antibody (FITC-coupled anti-rabbit immunoglobulin) or with secondary antibody alone (control). Immunofluorescence microscopy revealed that native RGS4 protein was localized to the cytoplasm and plasma membrane (Figure 1
, A and B).
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To determine the subcellular localization of transfected RGS4, the cDNA
encoding RGS4 was linked in-frame to the myc-1 epitope and
inserted into a mammalian expression vector (pCMV-RGS4-myc).
Cardiomyocytes were transfected with this construct, and
immunofluorescence microscopy was performed that
revealed that RGS4-myc protein was present chiefly in
the cytoplasm, with some found in the plasma membrane (Figure 1C
).
The level of RGS4 overexpression achieved by transfection with pCMV-RGS4-myc was determined by densitometric analysis of immunofluorescence microscopy images. The signal intensity obtained with anti-RGS4 antibody was compared in cells that were immunoreactive to anti–ß-galactosidase antibody (transfected cells) and those that were not immunoreactive to anti–ß-galactosidase antibody (nontransfected cells) and revealed that transfection with 1.5 µg pCMV-RGS4-myc resulted in a 2.5- to 3.2-fold increase in RGS4 protein levels (see "Methods").
Gene Induction Experiments
To determine whether RGS4 overexpression could inhibit signaling
by growth-promoting ligands, pCMV-RGS4-myc (or empty vector)
was triple-transfected with a CMV promoter–ß-galactosidase vector
and a promoter-reporter construct such as pANF-luc that has
ANF promoter sequence linked to cDNA encoding firefly luciferase. After
cells were stimulated with ligand, luciferase activity was measured and
normalized by ß-galactosidase activity (to account for variability in
transfection efficiency).
ANF was examined first because ligand stimulation of
cardiomyocytes results in robust induction of
ANF gene expression. In control cardiomyocytes
that were transfected with a CMV promoter-ß-galactosidase vector and
pANF-luc but not with pCMV-RGS4-myc, stimulation
with the -adrenergic ligand phenylephrine resulted in an
8- to 15-fold induction of ANF gene expression.
Phenylephrine-induced ANF gene expression
was inhibited in cells that were transfected with 1.5 µg or more
pCMV-RGS4-myc plasmid (Figure 2A).
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To determine whether the effect of RGS4-myc was specific to
its ability to deactivate G proteins, we transfected
cardiomyocytes with a construct encoding a point mutant
form of RGS4, N128A, which lacks GAP activity when tested in
vitro.31 Phenylephrine-induced
ANF gene expression was not reduced in
N128A-RGS4-myc cells compared with that in control cells
(Figure 2B).
In control cardiomyocytes that were transfected with a CMV
promoter–ß-galactosidase vector and pANF-luc but not with
pCMV-RGS4-myc, stimulation with a second ligand that signals
through heterotrimeric G proteins, endothelin-1, resulted in a 5- to
10-fold induction of ANF gene expression.
Endothelin-1–induced ANF expression was inhibited in cells
that were transfected with 1.5 µg pCMV-RGS4-myc.
ANF gene induction as measured by luciferase activity and
normalized by ß-galactosidase activity was reduced by 60% in
RGS4-myc cells (Figure 2C).
To control for nonspecific inhibitory effects of RGS4
overexpression, cardiomyocytes were also stimulated with
bFGF. In contrast to phenylephrine and endothelin-1, bFGF
does not signal through heterotrimeric G proteins but instead
activates intracellular signaling pathways, such as the MAP
kinase cascade, using the intrinsic tyrosine kinase activity of the
bFGF receptor.22 In control cardiomyocytes
that were transfected with a CMV promoter–ß-galactosidase vector and
pANF-luc but not with pCMV-RGS4-myc, stimulation
with bFGF resulted in a 3- to 4-fold induction of ANF gene
expression. As predicted, bFGF-induced ANF gene expression
was not blocked in cardiomyocytes that were transfected
with 1.5 µg pCMV-RGS4-myc (Figure 3A) or pCMV-N128A-RGS4-myc
(Figure 3B), demonstrating that the effect of
RGS4-myc was specific to a heterotrimeric G protein–coupled
pathway. Furthermore, treatment of cardiomyocytes with the
phorbol ester TPA resulted in ANF gene induction that was
not inhibited by overexpression of RGS4 (data not shown).
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The ability of RGS4 overexpression to block the expression of
MLC-2 was also examined. The MLC-2 gene encodes a
sarcomeric protein that is associated with cardiac
hypertrophy.16 MLC-2 gene
induction occurs as a result of activation of the MAP kinase and other
signaling pathways.13 14 In control
cardiomyocytes that were transfected with a CMV
promoter–ß-galactosidase vector and pANF-luc but not with
pCMV-RGS4-myc, stimulation with phenylephrine
resulted in a 3- to 4-fold induction in MLC-2 gene
expression as measured by luciferase activity normalized by
ß-galactosidase activity. Stimulation of control
cardiomyocytes with endothelin-1 or bFGF also resulted in a
3- to 4-fold induction of MLC-2 gene expression.
Phenylephrine- and endothelin-1–induced MLC-2
gene expression was inhibited in cardiomyocytes that were
transfected with 1.5 µg pCMV-RGS4-myc (Figure 4A, 4B). Transfection with 1.5 µg
pCMV-RGS4-myc did not inhibit bFGF-induced MLC-2
gene expression (Figure 4C).
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Analysis of Myofilament Organization and Cell Size
In cultured cardiomyocytes, mechanical stress and
ligands promote a hypertrophic response that includes the organization
of contractile proteins into sarcomeres. To test whether RGS4
overexpression could inhibit ligand-induced myofilament organization,
we examined actin polymerization by
immunofluorescence microscopy. Cardiomyocytes were
double-transfected with CMV promoter–ß-galactosidase vector and with
pCMV-RGS4-myc and were stimulated with the ligand
phenylephrine for 40 hours. Cells were fixed and
double-immunofluorescence microscopy was performed.
Transfected cells were identified by virtue of their ß-galactosidase
staining. Myofilament organization was assessed with the use of
rhodamine-tagged phalloidin. Phenylephrine-stimulated
myofilament organization was inhibited in cells that were transfected
with 1.5 µg pCMV-RGS4-myc compared with that in
nontransfected cells (Figure 5, A through
D).
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To determine whether RGS4-myc could also inhibit
ligand-induced increases in cell size, morphometric analysis of
cardiomyocytes was performed. Cardiomyocytes
double-transfected with CMV promoter-ß-galactosidase vector and with
1.5 µg pCMV-RGS4-myc were stimulated with the ligand
phenylephrine for 40 hours. Transfected cells were
identified by virtue of their ß-galactosidase staining.
Analysis of transfected and nontransfected cells with NIH Image
software revealed that the cell surface area of PE-stimulated
RGS4-myc transfected cells was significantly smaller
(P<0.0001) than that of PE-stimulated nontransfected cells
(Figure 6).
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Discussion |
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G proteins are thought to play an important role in the regulation of hypertrophic cardiac growth. RGS family members are GTPase-activating proteins for heterotrimeric G proteins of the Gi and Gq but not Gs families. We have demonstrated that overexpression of RGS4 can inhibit the ability of 2 ligands, phenylephrine and endothelin-1, to induce the cardiomyocyte hypertrophic growth program as assayed by the expression of marker genes, the organization of myofilaments, and by the analysis of cell size. Phenylephrine and endothelin-1 signal through heterotrimeric G proteins of the Gi and Gq families, respectively. Overexpression of RGS4 in cardiomyocytes does not inhibit the ability of bFGF, a ligand that does not signal through heterotrimeric G proteins, to increase the expression of the marker genes.
One assumption in this work is that RGS overexpression results in
deactivation of heterotrimeric G proteins. It has not been possible to
directly measure the activation state of heterotrimeric G proteins in
cells, for example, by determining the fraction of
Gq that is bound to GTP and not GDP. To
exclude the possibility that RGS4 inhibits endothelin-1 and
phenylephrine-mediated cardiomyocyte
hypertrophy by a mechanism separate from its ability to
promote GTPase activity, we performed 3 control experiments. In the
first we observed that overexpression of N128A-RGS4, a point mutant
form of RGS4 that lacks GAP activity, did not inhibit
phenylephrine-induced or endothelin-1–induced gene
induction. In the second we found that RGS4 overexpression did not
block bFGF-induced gene induction in cardiomyocytes.
Indeed, others have reported that bFGF does not signal through
heterotrimeric G proteins but instead activates the MAP kinase
cascade through a pathway that involves FRS2, Grb2, SOS, and
ras.22 In the third experiment we
observed that RGS4 overexpression did not block phorbol ester–induced
gene induction. Short-term administration of phorbol ester results in
the direct activation of protein kinase C, leading to activation of
Raf-1 and MAP kinase.32 These results
imply that RGS4 overexpression does not nonspecifically inhibit gene
induction in cardiomyocytes and also suggests that RGS4
activity is upstream of ras, Raf-1, and protein
kinase C.
To determine the role of RGS proteins in cardiomyocyte hypertrophy, we used the rat neonatal cardiomyocyte model. There are 3 caveats about the rat cardiomyocyte system that should be considered. First, cultured cardiomyocytes can be contaminated with cardiac fibroblasts. To address this issue, we (1) plated purified cardiomyocytes at low density, (2) used selective media, and (3) added 0.1 mmol/L BrdU to the cultures to inhibit nonmyocyte cell proliferation. Second, the transfection efficiency of cultured cardiomyocytes is often low (<10%) and can also vary significantly. To control for low or variable transfection efficiency, we used a ß-galactosidase expression vector under control of the human CMV promoter and measured ß-galactosidase activity in all transfected cells. In our experiments, luciferase data were always normalized by ß-galactosidase activity to control for variation in transfection efficiencies. Third, cultured cardiomyocytes may not be an accurate model of cardiac hypertrophy in live animals. It is clear that cultured homogenous isolated cells are not perfectly representative of an intact heart, which is a multilayered complex tissue with many cell types. However, there is considerable evidence that agents that promote hypertrophy in cultured cells, such as endothelin-1, angiotensin II, and mechanical stretch, also play a role in cardiac hypertrophy in intact animals. Confirmation of results obtained in transfected cardiomyocytes will have to be sought in transgenic animals.
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Acknowledgments |
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Dr Muslin was supported by grants from the Barnes-Jewish Hospital Foundation, the American Heart Association, and the National Institutes of Health. Dr Blumer is an Established Investigator of the American Heart Association.
Received April 29, 1998; revision received September 9, 1998; accepted September 15, 1998.
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