We showed before that in neonatal rat cardiac
myocytes partial inhibition of
Na+/K+-ATPase by nontoxic concentrations
of ouabain causes hypertrophic growth and transcriptional regulations
of genes that are markers of cardiac hypertrophy. In view of the
suggested roles of Ras and p42/44 mitogen-activated protein kinases
(MAPKs) as key mediators of cardiac hypertrophy, the aim of this work
was to explore their roles in ouabain-initiated signal pathways
regulating four growth-related genes of these myocytes,
i.e. those for c-Fos, skeletal
-actin, atrial
natriuretic factor, and the
3-subunit of
Na+/K+-ATPase. Ouabain caused rapid activations
of Ras and p42/44 MAPKs; the latter was sustained longer than 90 min.
Using high efficiency adenoviral-mediated expression of a
dominant-negative Ras mutant, and a specific inhibitor of MAPK kinase
(MEK), activation of Ras-Raf-MEK-p42/44 MAPK cascade by ouabain was
shown. The effects of the mutant Ras, an inhibitor of Ras
farnesylation, and the MEK inhibitor on ouabain-induced changes in
mRNAs of the four genes indicated that (a) skeletal
-actin induction was dependent on Ras but not on p42/44 MAPKs, (b)
3 repression was dependent on the
Ras-p42/44 MAPK cascade, and (c) induction of
c-fos or atrial natriuretic factor gene occurred partly
through the Ras-p42/44 MAPK cascade, and partly through pathways
independent of Ras and p42/44 MAPKs. All ouabain effects required
extracellular Ca2+, and were attenuated by a
Ca2+/calmodulin antagonist or a protein kinase C inhibitor.
The findings show that (a) signal pathways linked to
sarcolemmal Na+/K+-ATPase share early segments
involving Ca2+ and protein kinase C, but diverge into
multiple branches only some of which involve Ras, or p42/44 MAPKs, or
both; and (b) there are significant differences between
this network and the related gene regulatory pathways activated by
other hypertrophic stimuli, including those whose responses involve
increases in intracellular free Ca2+ through different
mechanisms.
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INTRODUCTION |
Na+/K+-ATPase catalyzes the coupled active
transport of Na+ and K+ across the plasma
membranes of most mammalian cells (1). In the myocardium,
Na+/K+-ATPase also regulates contractility; the
partial inhibition of the myocardial enzyme by ouabain and related
digitalis drugs causes a small increase in intracellular
Na+, which in turn affects the sarcolemmal
Na+/Ca2+ exchanger, leading to a significant
increase in intracellular Ca2+ and in the force of
contraction (2). This positive inotropic action is the basis of the
continued use of digitalis drugs in the treatment of congestive heart
failure (3, 4). Recently, using cultured neonatal rat cardiac myocytes,
we showed (5-7) that the same nontoxic concentrations of ouabain that
cause partial inhibition of sarcolemmal
Na+/K+-ATPase and increased intracellular
Ca2+, also stimulate the hypertrophic growth of these
myocytes, and lead to transcriptional regulation of several genes that
have been implicated as markers of cardiac hypertrophy. Clearly, the altered activity of Na+/K+-ATPase by digitalis
drugs must now be considered as a potential signal for hypertrophic
growth along with other hormonal, mechanical, and pathological stimuli
of cardiac hypertrophy (8). Cardiac hypertrophy is not only a
beneficial adaptive response to increased workload, but also a prelude
to the development of heart failure (9). Hence, there is considerable
interest in the potential hypertrophic effects of drugs that are widely
used in the treatment of heart failure.
Based on studies from both in vivo and in vitro
models of hypertrophy, it is evident that transcriptional regulations
of some early-response protooncogenes and late-response fetal genes are associated with myocyte hypertrophy, and that these genes are regulated
in distinctively different patterns in response to different hypertrophic stimuli (8-11). While the inductions of these
growth-related genes are not sufficient for hypertrophic growth of
myocytes (12-15), it is clear that for any hypertrophic stimulus, the
mapping of the signal transduction pathways involved in the regulation
of growth-related genes is necessary for the definition of the
hypertrophic phenotype induced by that stimulus. Therefore, the aim of
this work was the continuation of our recent efforts to characterize the molecular mechanisms of the linkage between the sarcolemmal Na+/K+-ATPase and the expressions of several
growth-related genes of cultured neonatal rat cardiac myocytes.
Specifically, in view of the suggested roles of Ras (16-18) and p42/44
MAPKs1 (19-22) in the
development of cardiac hypertrophic phenotype, we wished to assess the
possible involvements of Ras and these MAPKs in the ouabain-initiated
signal transduction pathways of four genes that we had shown to be
transcriptionally regulated in the course of ouabain-induced
hypertrophy of cardiac myocytes, i.e. c-fos and
the genes for ANF, skACT, and the
3-subunit of Na+/K+-ATPase (5-7). Our findings indicate
that while the four ouabain-initiated pathways share an early segment
involving Ca2+, calmodulin, and PKC, they subsequently
branch out into multiple Ras-dependent and Ras-independent
segments only some of which are also dependent on p42/44 MAPKs.
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EXPERIMENTAL PROCEDURES |
Materials--
Chemicals of the highest purity available were
purchased from Sigma. TRI reagent for RNA isolation was from Molecular
Research Center, Inc. (Cincinnati, OH). Radionucleotides
(32P-labeled, about 3,000 Ci/mmol) and
[32P]Pi were from NEN Life Science Products.
Rabbit Anti-ACTIVE MAPK polyclonal antibody and anti-p42/44 antibodies
were obtained from Promega (Madison, WI) and New England Biolabs
(Beverly, MA), respectively. An anti-Ha-Ras monoclonal antibody and
protein G plus agarose were obtained from Santa Cruz Biotechnology,
Inc. (Santa Cruz, CA). All protein kinase inhibitors were purchased
from Calbiochem (San Diego, CA).
Cell Preparation and Culture--
Neonatal ventricular myocytes
were prepared and cultured as described in our previous work (5-7).
Briefly, myocytes were isolated from ventricles of 1-day-old
Sprague-Dawley rats, and purified by centrifugation on Percoll
gradients. Myocytes were then cultured in a medium containing 4 parts
of Dulbecco's modified Eagle's medium and 1 part Medium 199 (Life
Technologies, Inc.), penicillin (100 units/ml), streptomycin (100 µg/ml), and 10% fetal bovine serum. After 24 h of incubation at
37 °C in humidified air with 5% CO2, medium was changed
to one with the same composition as above, but without the serum. All
experiments were performed after another 48 h of incubation under
serum-free conditions. Incubations in Ca2+-free media were
performed as described previously (5). These cultures contain more than
95% myocytes as assessed by immunofluorescence staining with a myosin
heavy chain antibody.
Northern Blot--
Northern blot was performed as described
previously (5, 6). Routinely, about 20 µg of total RNA was subjected
to gel electrophoresis, transferred to a Nytran membrane,
UV-immobilized, and hybridized to 32P-labeled probes.
Autoradiograms obtained at
70 °C were scanned with a Bio-Rad
densitometer. Multiple exposures were analyzed to assure that the
signals are within the linear range of the film. The relative amount of
RNA in each sample was normalized to that of GAPDH mRNA to correct
for differences in sample loading and transfer.
Measurement of Phosphorylation and In-gel Assay of p42/44
MAPKs--
Activation of p42/44 MAPKs in cultured myocytes was
determined by both in-gel kinase assay and Western blot using a rabbit polyclonal antibody raised against dually phosphorylated p42/44 MAPKs
(23, 24). In brief, after cells were exposed to ouabain, reaction was
terminated by the replacement of medium with 200 µl of ice-cold lysis
buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM NaF, 1 mM Na3VO4, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 50 mM tetrasodium pyrophosphate, 10 nM okadaic
acid, 1% Triton X-100, 0.25% sodium deoxycholate, 10 µg/ml
aprotinin, and 10 µg/ml leupeptin). Protein content was measured by
the Lowry method (25) using bovine serum albumin as standard. For
Western blot analysis, cell lysates (30 µg/lane) were
electrophoresized on 10% SDS-polyacrylamide gels, and transferred to a
nitrocellulose membrane. The membranes were probed with Anti-ACTIVE
MAPK polyclonal antibody, which detects p42/44 MAPKs only when they are
activated by phosphorylation at Thr-202 and Tyr-204. To ensure equal
loading and protein transfer, duplicate blots were performed for the
same samples and probed with a polyclonal antibody recognizing both
phosphorylated and non-phosphorylated p42/44 MAPKs. These membranes
were developed with a secondary anti-rabbit antibody as we previously
described (7). For in-gel assay, cell lysates (30 µg/lane) were
resolved in 10% SDS-polyacrylamide gels containing 0.5 mg/ml myelin
basic protein. MAPKs in the gels were denatured in 6 M
guanidine-HCl and renatured in 50 mM Tris-HCl (pH 8.0)
containing 0.05% Triton X-100 and 5 mM 2-mercaptoethanol.
The kinase activities were assayed by incubation of these gels with
[
-32P]ATP. The gels were then extensively washed,
dried, and subjected to autoradiography. Both Western blots and
autoradiograms were scanned with a Bio-Rad densitometer to quantitate
MAPK signals.
Analysis of GTP and GDP-bound Ras--
Measurement of guanine
nucleotide-bound Ras was performed as described previously (26, 27).
Cells in 60-mm dishes were prelabeled with 0.4 mCi of
32Pi for 18 h in phosphate-free
Dulbecco's modified Eagle's medium. After exposure to 100 µM ouabain for various times, cells were washed twice
with ice-cold phosphate-buffered saline, and lysed in 0.5 ml of lysing
buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 20 mM MgCl2, 1 mM
Na3VO4, 1 mM phenylmethylsulfonyl
fluoride, 1% Triton X-100, 10 µg/ml aprotinin, and 10 µg/ml
leupeptin). Cell lysates were centrifuged for 5 min at 2,000 × g, and the supernatant was incubated with an anti-Ha-Ras
monoclonal antibody for 1 h at 4 °C. The immune complexes were
then precipitated with protein G plus agarose overnight at 4 °C. The
precipitates were washed four times with the lysing buffer and three
times with washing buffer (Tris-HCl, pH 7.4, 20 mM
MgCl2, and 150 mM NaCl). The Ras-bound GDP and
GTP were then eluted in 20 µl of elution buffer (20 mM
Tris-HCl, pH 7.4, 20 mM EDTA, 2% SDS, 0.5 mM
GDP, and 0.5 mM GTP). Eluted GDP and GTP were separated on
a polyethyleneimine-cellulose thin layer plate and developed with 0.75 M KH2PO4 (pH 3.4). Labeled GDP and
GTP were visualized by autoradiography. GDP and GTP fractions were then
cut out from the plate and quantitated by scintillation counting. Ras
activity is expressed as a ratio of GTP/(GDP + GTP).
Preparation of Replication-defective Adenovirus Asn17
Ras and Adenovirus Infection of Cardiac Myocytes--
A
replication-defective adenovirus expressing dominant negative
Asn17 Ras was generated as described previously (28). Virus
was amplified in human kidney 293 cells, and the viral particles were
purified from 293 cell lysates by cesium chloride gradient
ultracentrifugation then desalted by dialysis (29). The concentration
of recombinant adenovirus was determined based on the absorbance at 260 nm where 1 optical density unit corresponds to 1012
particles/ml. An identical adenovirus containing the
-galactosidase gene instead of the Asn17 Ras was used as a virus control.
In three independent infection experiments with different
concentrations of adenovirus-
-galactosidase, the percentages of
cardiac myocytes expressing
-galactosidase after 12 h of
infection, as determined by histochemical staining, were 81 ± 5%
(100 viral particles/cell) and 96 ± 3% (1,000 viral particles/cell).
Statistics--
Data are given as mean ± S.E. Statistical
analysis was performed using Student's t test, and
significance was accepted at p < 0.05.
 |
RESULTS |
Ouabain Activates p42/44 MAPKs in Cardiac
Myocytes--
Involvement of p42/44 MAPK isoforms (ERK1 and ERK2) in
the development of cardiac myocyte hypertrophy has been suggested, and
activations of these MAPKs by several cardiac hypertrophic stimuli such
as phenylephrine, endothelin, and PMA have been demonstrated (19-22).
Since our previous studies suggested that p42/44 MAPKs may participate
in ouabain-induced down-regulation of the
3 subunit of
Na/K-ATPase in cardiac myocytes (7), we wished to determine if these
kinases are indeed activated when myocytes are exposed to ouabain
concentrations that cause partial inhibition of
Na+/K+-ATPase. Myocytes were exposed to 100 µM ouabain for various times, and the levels of
phosphorylated and activated p42/44 MAPKs were measured by Western blot
analysis. As shown in Fig. 1A,
ouabain increased phosphorylation levels of both p42 and p44 in a
time-dependent manner. Because p42 Western blot signals
were much stronger than those of p44, ouabain-induced increase in p42
phosphorylation was quantitated as shown in Fig. 1B. A
significant activation of p42 MAPK was observed after 5 min of
exposure, reached maximal value after 15 min, and lasted for at least
90 min. Limited quantitations were also performed for p44 in several
overdeveloped membranes, and similar activation patterns were noted
(data not shown). When concentration-dependent changes in
response to ouabain were measured, significant activations of both p42
and p44 MAPKs were observed with ouabain concentrations as low as 10 µM (Fig. 2). To confirm if
ouabain-induced increases in MAPK phosphorylation are correlated with
increase in MAPK activity, extracts from myocytes treated with 100 µM ouabain for 15 min were also analyzed using the in-gel kinase assay (see "Experimental Procedures"). In three independent experiments, ouabain caused a 2.6 ± 0.5-fold increase in p44 and a 3.2 ± 0.4-fold increase in p42 activities.

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Fig. 1.
Time course of the effects of ouabain on
p42/44 MAPKs. Cells were treated with 100 µM ouabain
for various times and assayed for p42/44 MAPKs as described under
"Experimental Procedures." A, a representative Western
blot. The top panel was detected by an antibody specific for
dual phosphorylated p42/44, and the bottom panel was
detected by an antibody recognizing total p42/44 MAPKs, showing an
equal loading and protein transfer among different lanes. B,
combined data from four independent experiments. Phosphorylated p42
values were normalized against the total p42 signal and expressed as
mean ± S.E.
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Fig. 2.
Dose-dependent effects of ouabain
on p42/44 MAPKs. Cells were treated with various concentrations of
ouabain for 15 min, and assayed for p42/44 MAPK phosphorylation as in
Fig. 1. A representative Western blot of three independent experiments
is shown. Lane 1, control; lane 2, 10 µM ouabain; lane 3, 100 µM
ouabain.
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Phosphorylation and activation of p42/44 MAPKs are catalyzed by MEK,
and PD98059 has been shown to be a highly specific inhibitor of MEK
(30). To verify the role of MEK in ouabain-induced activation of p42/44
MAPKs, in experiments similar to those of Figs. 1 and 2, myocytes
pretreated for 15 min with 10 µM PD98059 and control myocytes were exposed to 100 µM ouabain for 15 min, and
assayed for MAPK phosphorylation. In three separate experiments,
PD98059 caused 93 ± 6% suppression of ouabain-induced
phosphorylation of p42/44 MAPKs.
Ouabain Stimulates p42/44 MAPKs through a Ras-dependent
Pathway--
Activations of MEK and p42/44 MAPKs may occur through the
Ras-/Raf-pathway or Ras-independent pathways (31). To test whether Ras
is involved in ouabain-induced activation of MAPK, cells were infected
with an adenovirus expressing an Asn17 dominant-negative
mutant of Ras for 12 h, washed, then exposed to either ouabain or
PMA for 15 min. Adenovirus
-galactosidase-infected myocytes served
as control. As depicted in Fig. 3,
expressing dominant negative Ras blocked ouabain-induced but not
PMA-induced, activation of p42 MAPK, indicating that ouabain signals
through Ras in contrast to PMA, which is known to use a Ras-independent pathway in activation of MAPKs (32, 33).

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Fig. 3.
Effects of expression of dominant negative
Ras on ouabain and PMA-induced p42 MAPK activation. Cells were
transduced either with the adenovirus expressing an Asn17
dominant-negative mutant of Ras or with the control virus containing
-galactosidase gene as indicated at a dose of 1,000 particles/cell.
After 12 h of incubation, both control and virus-infected cells
were washed, then exposed to either 100 µM ouabain or 100 nM PMA for 15 min, and assayed for p42 MAPK phosphorylation
as in Fig. 2. Values are mean ± S.E. of three experiments.
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That Ras is indeed activated when myocytes are exposed to ouabain was
shown in experiments of Fig. 4,
indicating that 100 µM ouabain induced a rapid
accumulation of GTP-bound Ras, which reached maximum in 5 min and
returned to basal levels after 60 min. This time course of
ouabain-induced Ras activation correlates well with the time course of
MAPK activation by ouabain (Fig. 1).

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Fig. 4.
Time course of ouabain-induced Ras
activation. Cells were labeled with
[32P]Pi for 18 h, then exposed to 100 µM ouabain for various times. Ras was immunoprecipitated
with an anti-Ha-Ras monoclonal antibody, and Ras-bound GTP and GDP were
separated as described under "Experimental Procedures."
A, a representative autoradiogram. B, combined
data from four independent experiments. Values are mean ± S.E.
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Different Roles of MAPKs in Ouabain-induced Regulations of the
Growth-related Genes--
We showed before (7) that the MEK inhibitor
PD98059 blocked ouabain-induced repression of
3
mRNA, but had no effect on ouabain-induced increase in skACT
mRNA. To assess the roles of MEK-p42/44 MAPKs in ouabain-activated
pathways regulating c-fos and ANF gene, myocytes that were
pretreated with PD98059 were exposed to 100 µM ouabain
and assayed for appropriate mRNAs. The results, along with the
effects of PD98059 on
3 and skACT mRNA, are
presented in Fig. 5. Clearly, under the
same condition where PD98059 has no influence on ouabain's effect on
skACT, the MEK inhibitor causes complete blockade of the ouabain effect
on
3 and only partial blockade of the ouabain effects on
ANF and c-fos. It is important to note that 10 µM PD98059 used in experiments of Fig. 5 causes near
complete inhibition of myocyte MAPK phosphorylation (Ref. 23 and the
results presented above); and that concentrations of PD98059 higher
than 10 µM caused no further effects than those shown in
Fig. 5 (data not shown).

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Fig. 5.
Effects of PD98059 on ouabain-induced
c-fos, skACT, 3, and ANF expression.
Myocytes were pretreated with 10 µM PD98059. After 15 min
of incubation, both control and PD98059-treated cells were exposed to
100 µM ouabain for 45 min, then assayed for
c-fos mRNA. In another set of experiments, cells were
treated with 100 µM ouabain for 12 h, then assayed
for skACT, ANF, and 3 mRNA. Ouabain treatment times
were chosen to obtain maximal effects on the mRNAs of the four
genes (5-7). Assays were performed as described under "Experimental
Procedures." mRNA values were normalized to those of
corresponding GAPDH measured on the same blot and expressed relative to
a control value of one. The values are mean ± S.E. of three
experiments. A, representative Northern blots of
c-fos and ANF mRNAs. B, effects of ouabain on
c-fos and skACT. C, effects of ouabain on ANF and
3.
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Different Roles of Ras in Ouabain-induced Regulations of the
Growth-related Genes--
Since ouabain activation of p42/44 MAPKs
seems to be through Ras/Raf/MEK/MAPK sequence (Fig. 3), and since some
of the ouabain-activated pathways regulating the four ouabain-regulated
genes do not involve p42/44 MAPK activations (Fig. 5), it was of
interest to explore the role of Ras in the MAPK-dependent
and MAPK-independent pathways of the ouabain-regulated genes. Myocytes
were infected with the adenovirus containing the dominant-negative Ras,
exposed to ouabain, and assayed for the appropriate mRNAs. The
results summarized in Fig. 6 showed that
the expression of dominant-negative Ras caused (a) complete
blockade of the ouabain-induced increase in skACT mRNA,
(b) complete reversal of the ouabain-induced decrease in
3 mRNA, and (c) partial blockade of the
ouabain-induced increase in ANF mRNA. Quantitation of the results
of similar experiments on ouabain-induced increase in c-fos
mRNA were problematic, since the control
-galactosidase vector
had significant effects on c-fos expression. However,
neither for c-fos nor in the case of ANF (Fig.
6C) was it possible to approach complete blockade of the
ouabain effect by using higher concentrations of the virus containing
the dominant-negative Ras (not shown).

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Fig. 6.
Expression of dominant negative Ras
suppresses the effects of ouabain on late response genes. Cells
were transduced either with the adenovirus expressing an
Asn17 dominant-negative mutant of Ras or with the control
virus containing -galactosidase gene as indicated at a dose of 1,000 particles/cell. Both control and virus-infected cells were then washed
and treated with 100 µM ouabain for additional 12 h,
and assayed for skACT (A), 3 (B),
ANF (C), and GAPDH mRNAs as in Fig. 5. Ouabain treatment
times were chosen to obtain maximal effects on the indicated mRNA
(6, 7). Values are mean ± S.E. of four experiments.
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To confirm the role of Ras in ouabain-induced regulation of the cardiac
genes, we used
-hydroxyfarnesylphosphoric acid, a cell-permeable
farnesyltransferase inhibitor that is known to block Ras farnesylation,
prevent its association with the plasma membrane, and inhibit Ras
signaling (34, 35). As depicted in Fig.
7, when myocytes were pretreated with
this inhibitor, a dose-dependent suppression of
ouabain-induced skACT expression was noted. This inhibitor also caused
significant reversal of ouabain-induced up-regulation of ANF and
down-regulation of
3 mRNAs (data not shown).

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Fig. 7.
Inhibition of Ras farnesylation represses
ouabain-induced skACT expression. Cells were pretreated with
different concentrations of -hydroxyfarnesylphosphoric acid for
18 h. Both control and inhibitor-treated cells were then exposed
to 100 µM ouabain for 12 h, and assayed for skACT
and GAPDH mRNAs as in Fig. 5. Values are mean ± S.E. of three
experiments.
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Ouabain Effects on p42/44 MAPKs and the Growth-related Genes Are
Dependent on Extracellular Ca2+, Calmodulin, and
PKC--
We demonstrated previously (5-7) that the presence of
extracellular Ca2+ was required for ouabain-induced
regulations of c-fos, skACT, and
3 subunit
genes in myocytes. Experiments, the results of which are summarized in
Fig. 8, showed that extracellular
Ca2+ was also necessary for activation of p42/44 MAPKs and
for increase of ANF mRNA caused by ouabain.

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Fig. 8.
Effects of extracellular Ca2+ on
ouabain-induced ANF expression and p42 MAPK phosphorylation. Cells
were treated with 100 µM ouabain either in the control
medium or in the Ca2+-free medium for either 15 min
(MAPK) or 12 h (ANF), then assayed for p42
MAPK phosphorylation and ANF mRNA respectively. Ouabain treatment
times were chosen to obtain maximal effects on p42 MAPK (Fig. 1) and on
ANF induction (6). The assays were performed as in Fig. 1 and Fig.
5.
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Our previous studies suggested the involvements of calmodulin and PKC
in the ouabain-induced pathways regulating the expressions of skACT,
and the
3 subunit (6, 7). Experiments of Fig. 9 showed that membrane permeable
Ca2+/calmodulin antagonist W-7, and PKC inhibitor H-7, also
antagonized activation of MAPKs, and increased expressions of
c-fos and ANF by ouabain.

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Fig. 9.
Effects of protein kinase inhibitors on
ouabain-induced p42 MAPK phosphorylation and expressions of
c-fos and ANF mRNAs. Cells were pretreated with
different protein kinase inhibitors as indicated for 15 min, then
exposed to 100 µM ouabain for 15 min, and assayed for p42
MAPK phosphorylation. In the other two sets of experiments, the
inhibitor-pretreated cells were exposed to ouabain for either 45 min or
12 h, then assayed for c-fos and ANF mRNAs,
respectively. Ouabain treatment times were chosen to obtain maximal
effects on the indicated mRNAs (5, 6). Values are mean ± S.E.
of three experiments.
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Because H-7 also inhibits protein kinase A, a more selective inhibitor
of this kinase (HA1004) was also used in experiments of Fig. 9, as it
was used in our previous studies (6, 7). HA1004 (50 µM)
had no significant effect on ouabain-induced increases in
c-fos and ANF mRNAs, and activations of p42/44 MAPKs
(data not shown); supporting the assumption that H-7 effects noted in Fig. 9 are due to PKC inhibition. The combined results of Figs. 8 and 9
and our previous data (5-7) suggest that increased Ca2+
influx, calmodulin, and PKC affect the signal pathways of the four
ouabain-regulated genes upstream of the Ras/Raf/MEK/MAPK sequence.
Myocyte Viability--
We showed before (5) that myocyte
viability, as measured by lactate dehydrogenase release, was not
affected by ouabain concentrations used here. In similar experiment, we
found that myocyte viability was also not affected significantly when
10 µM PD98059, 10 µM W-7, or 50 µM H-7 was added to the culture medium in addition to
ouabain, and incubations were carried out for the longest indicated
periods (data not shown). In agreement with others (55), we also found
that adenoviral infection of these neonatal myocytes under the
indicated conditions had no significant effect on myocyte viability
(data not shown).
 |
DISCUSSION |
A number of physiological, pathological, and pharmacological
stimuli are known to cause cardiac myocyte hypertrophy (8). To
determine how these stimuli work in concert to regulate cardiac growth,
remodeling, and failure, it is necessary to define the signal
transduction pathways that are activated by the various stimuli, and to
clarify the mechanisms of cross-talk among these pathways. In this
context, and in view of our recent discovery of the hypertrophic
effects of ouabain on cardiac myocytes (5, 6), the primary objectives
of this work were to determine if partial inhibition of cardiac myocyte
Na+/K+-ATPase leads to activations of Ras and
p42/44 MAPKs, and to assess the roles of any such activations in the
signal pathways that link the sarcolemmal
Na+/K+-ATPase to the expression of several
growth-related genes of these myocytes. In all experiments, we used
ouabain at concentrations of 100 µM or less, which are
known to cause less than 50% inhibition of
Na+/K+-ATPase, and no overt toxicity, in
neonatal rat myocytes (5). Under these conditions that mimic those
under which ouabain causes its positive inotropic effect on the heart
(2), our findings clearly show that Ras and the MAPKs are rapidly
activated, and that these activations are within some, but not all,
gene regulatory pathways that are initiated by
Na+/K+-ATPase inhibition.
Multiplicity of Ouabain-initiated Gene Regulatory
Pathways--
Based on our data, the following conclusions, as
summarized in Fig. 10, emerge about the
roles of Ras and p42/44 MAPKs in the pathways that link the activity of
cardiac Na+/K+-ATPase to the transcriptions of
the four genes that we have studied here.

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Fig. 10.
Schematic representation showing that
multiple signal transduction pathways link
Na+/K+-ATPase to ouabain-specific regulation of
growth-related genes in cardiac myocytes.
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The case of the
3 subunit of
Na+/K+-ATPase is the most straightforward.
Since ouabain-stimulated activations of the p42/44 MAPKs are entirely
dependent on Ras (Fig. 3), and since ouabain-induced repression of
3 is completely reversed by blockade of Ras (Fig. 6B) or by MEK inhibition (Fig. 5B), it is clear
that repression of
3 by ouabain must proceed through the
well established Ras-Raf-MEK-p42/44 MAPK sequence (Fig. 10). The
inductions of c-fos and ANF gene by ouabain are similar in
that both are partially antagonized by Ras blockade or by MEK
inhibition (Figs. 5 (A and B) and 6C;
see "Results"), indicating the existence of at least two distinct branches leading to the regulation of each of these two genes: one
involving the Ras-Raf-MEK-p42/44 MAPKs sequence, and the other independent of Ras and these MAPKs (Fig. 10). Finally, since the induction of skACT by ouabain is completely prevented by the blockade of Ras (Fig. 6A), but not affected at all by MEK inhibition
(Fig. 5A), it must involve a Ras-dependent
pathway that is different from the Ras-Raf-MEK-p42/44 MAPKs sequence
(Fig. 10). The existence of such Ras-dependent pathways,
e.g. through Ras-MEK kinase-c-Jun N-terminal kinase, are
well established (8, 27, 31), and their possible involvement in skACT
induction by ouabain is being explored.
Further information regarding the signal pathways initiated by the
partial inhibition of cardiac Na+/K+-ATPase is
provided by our findings on the roles of Ca2+, calmodulin,
and PKC. The ouabain-induced regulations of the four genes studied
here, and the ouabain-induced activations of p42/44 MAPKs, are totally
dependent on extracellular Ca2+ (Fig. 8 and Refs. 5-7),
and all are prevented by a calmodulin antagonist or a PKC inhibitor
(Fig. 9 and Refs. 6 and 7). The most economical way of explaining these
data is to assume that each of these three factors (increased influx of
Ca2+, calmodulin, and PKC) has a single locus of action
that is shared by all ouabain-stimulated pathways and, therefore, must
be upstream of Ras in the Ras-dependent branches (Fig. 10).
The mechanism of Ras activation resulting from the inhibition of
Na+/K+-ATPase is not known. However, its
dependence on Ca2+/calmodulin, as depicted in Fig. 10,
provides a lead for the future clarification of this issue; the
Ca2+/calmodulin-dependent activation of Ras by
signals that do not directly interact with receptor tyrosine kinases,
but activate cytosolic tyrosine kinases, has been noted in cells other
than cardiac myocytes (24, 36).
If Ca2+/calmodulin and PKC act upstream of Ras, as
indicated in Fig. 10, the question may arise as to whether
ouabain-induced increase in intracellular free Ca2+, and
the postulated ouabain-induced PKC activation, are fast enough to be
consistent with the noted rapid activations of Ras and p42/44 MAPKs
(Figs. 1 and 4). There is ample prior evidence to show that, in
cultured chick cardiac myocytes, inotropic concentrations of ouabain
increase intracellular free Ca2+, reaching steady state
maximal levels within 5-6 min (Ref. 56 and references therein); we
have confirmed this using the myocyte preparation used
here.2 We know of no
published data on ouabain-induced activation of PKC, and its time
course, in cultured cardiac myocytes; however, in rabbit papillary
muscle, activation of PKC has been suggested to occur within 0.5-7 min
after the addition of ouabain (57). Regarding the roles of
Ca2+, calmodulin, and PKC, it is also important to note
that their additional downstream effects within the branched pathways
of Fig. 10 cannot be ruled out. For example, c-fos
transcription in cells other than myocytes is known to be regulated by
a number of downstream mechanisms involving Ca2+ or PKC
(37, 38). The existence of similar downstream effects within
Ras-dependent and -independent branches of Fig. 10 needs to
be explored.
Specificities of the Ouabain-initiated Gene Regulatory
Pathways--
The growth-related genes of the neonatal cardiac
myocytes, including the four we have studied here, are regulated by a
number of hypertrophic stimuli. Extensive previous studies on the
signal pathways that are activated in cardiac myocytes by well
established hypertrophic stimuli (e.g. phenylephrine,
angiotensin II, and mechanical stretch) have clearly shown the
existence of a complex network of pathways with stimulus-specific
segments and segments that are shared by different stimuli (8, 27, 33,
39, 40). The emerging information on the pathways that are activated by
ouabain (Fig. 10) also point to ouabain-specific effects within the
same over-all network. Although it is not practical to do a thorough
analysis of the interrelation of the ouabain-regulated pathways and
those of other stimuli, it is instructive to consider similarities and
differences between some of the ouabain effects noted here and the
related effects of other stimuli.
The p42/44 MAPKs of cardiac myocytes have been suggested to be key
mediators of cardiac hypertrophy by several studies (19-22); as is the
case for ouabain-induced pathways, recent work has established the role
of these kinases in some, but not all, signal transduction pathways
that are activated by other stimuli (33, 40-42). It is appropriate,
therefore, to see how ouabain effects on cardiac p42/44 MAPKs compare
with those of other stimuli. Ouabain causes rapid activation of p42/44
MAPKs, which is sustained for at least 90 min (Fig. 1), and is totally
dependent on extracellular Ca2+ (Fig. 8) and on Ras (Fig.
3). In similar myocyte preparations, activations of p42/44 MAPKs have
been noted by hypertrophic stimuli phenylephrine (23, 43),
isoproterenol (43), phorbol esters (43, 44), fibroblast growth factors
(44), endothelin (23, 44), angiotensin II (33, 45), mechanical stretch
(26, 42, 46), and by a number of receptor agonists that do not cause
hypertrophy (23). Unlike ouabain, however, all but one of the above
stimuli cause rapid but transient activation levels that return to
basal or lower than basal levels within 30-60 min (23, 26, 43-46).
Activation by phorbol esters has a time course similar to that of
ouabain (43-45); however, unlike ouabain activation, activation by
phorbol esters in these myocytes is not dependent on Ras (Fig. 3). The
importance of sustained activations of MAPKs in relation to their gene
regulatory actions has been noted (47, 48). The potential significance
of differences between the activation time courses of ouabain and the
other stimuli remains to be determined.
As in the case of ouabain-induced activation of p42/44 MAPKs,
extracellular Ca2+ is required for activations by
isoproterenol and angiotensin II (43, 45); however, activation by
phorbol esters, endothelin, fibroblast growth factor, and phenylephrine
are not dependent on extracellular Ca2+ (43). Additions of
Ca2+ ionophores to myocytes in Ca2+-containing
media also cause rapid activations of p42/44 MAPKs (43, 45); however,
these activations are also quite short-lived (45). Most interestingly,
electrical pacing which also causes cardiac myocyte hypertrophy and
increase in intracellular Ca2+, does not cause activations
of p42/44 MAPKs (49, 50). Taken together, the above clearly suggest
that increases in intracellular Ca2+ achieved by ouabain
and by other means have different effects on p42/44 MAPKs, lending
further support to our previous proposal (7) that in cardiac myocytes,
as in other cells (51), different intracellular pools of
Ca2+ may have different effects within the signal
pathways.
Other noteworthy differences between the ouabain effects presented here
and those of other stimuli are related to the skACT gene. Although this
fetal gene is induced by a large number of cardiac hypertrophic stimuli
(8), the signal pathways that lead to its induction have not been
studied as extensively as those of ANF induction. Nevertheless, the
available data point to significant ouabain selectivity within multiple
pathways of skACT induction. Our data clearly show that induction of
skACT by ouabain is totally Ras-dependent, but independent
of MEK-p42/44 MAPK activations (Figs. 5, 6, and 10). In similar
preparations of cardiac myocytes, however, stimulation of skACT
promoter activity by MEK and p42-MAPK has been shown in one study (21),
and other studies have failed to demonstrate the involvement of Ras in
transforming growth factor-
-induced regulation of skACT gene (52,
53). Interestingly, these latter studies indicated a "global" role of Ras in the basal expressions of a variety of cardiac myocyte genes.
With the use of the adenoviral vector for the high efficiency expression of the Ras mutant, and under the indicated experimental conditions, we see no evidence of such a global effect of Ras. The
phenylephrine-induced regulation of skACT is dependent on PKC (54), as
is the ouabain-induced regulation of skACT (6). Other similarities or
differences between the effects of ouabain and other stimuli on the
pathways regulating skACT expression remain to be explored.
In summary, our present and earlier findings (5-7) show that partial
inhibition of cardiac sarcolemmal
Na+/K+-ATPase, within the limits that are
achieved by the positive inotropic concentrations of digitalis drugs,
induces a distinct pattern of hypertrophy and gene regulation through
multiple pathways that are related but not identical to the network of
pathways that are activated by other hypertrophic stimuli. Studies on
the further characterization of the gene regulatory mechanisms that are
linked to cardiac Na+/K+-ATPase, and on the
mechanisms of interactions between these and the related pathways
activated by other physiological and pathological hypertrophic stimuli
are in progress.