(Received for publication, November 20, 1995; and in revised form, January 31, 1996)
From the
Exposure of neonatal rat cardiac myocytes to ouabain
concentrations that caused partial inhibition of
Na/K
-ATPase but no loss of viability,
increased c-fos and c-jun mRNAs and the transcription
factor AP-1. The increased mRNAs were proportional to the extent of
inhibition of Na
/K
-ATPase and the
resulting rise in steady state intracellular Ca
concentration. The rapid and sustained increase of c-fos mRNA
was shown to be due to increased transcriptional rate. Induction of
c-fos by ouabain was prevented when either extracellular or
intracellular Ca
was lowered and was attenuated by
pretreatment of myocytes with a phorbol ester under conditions known to
down-regulate protein kinase C. Exposure to ouabain for 24-48 h
also increased total transcriptional activity and protein content of
myocytes. The findings suggest that the same signal responsible for the
positive inotropic action of ouabain, i.e. net influx of
Ca
caused by partial inhibition of
Na
/K
-ATPase, also initiates the rapid
protein kinase C-dependent inductions of the early-response genes, the
subsequent regulations of other cardiac genes by the resulting
transcription factors, and stimulation of myocyte growth. Whether these
hitherto unrecognized effects of cardiac glycosides are obtained in the
intact heart and their relevance to the therapeutic uses of these drugs
remain to be determined.
Ouabain and related cardiac glycosides are highly specific
inhibitors of Na/K
-ATPase. This
enzyme (the sodium pump) catalyzes the coupled active transport of
Na
and K
across the plasma membranes
of most animal cells (1) . It is now well established that the
positive inotropic effect of a cardiac glycoside on the myocardium is
due to the partial inhibition of the cardiac
Na
/K
-ATPase, causing a small increase
in intracellular Na
, which in turn affects the
sarcolemmal Na
/Ca
exchanger, leading
to an increase in intracellular Ca
and in the force
of contraction(2, 3, 4) . This effect on
cardiac contractility is the basis of the major therapeutic use of
these drugs in the treatment of congestive heart
failure(2, 3, 4) . There is also evidence to
suggest that ouabain or related cardiac glycoside-like substances may
be paracrine hormones in higher animals(5) .
In several cell
types other than cardiac myocytes, inhibition of
Na/K
-ATPase, either by ouabain or by
low extracellular K
, has been shown to affect the
expression of proto-oncogenes c-fos and
c-jun(6, 7, 8) . Because the
inductions of such early-response genes have been implicated in cardiac
growth and hypertrophy(9, 10) , and since the
hypertrophied failing heart is the primary target of therapy with
cardiac glycosides, it was of interest to explore the effects of these
drugs on the expressions of cardiac early-response genes. Here, we
report the results of our initial studies in this direction, using the
primary cultures of neonatal rat cardiac myocytes as a model.
Neonatal ventricular myocytes were prepared and cultured as
described before(11, 12) . Briefly, myocytes were
isolated from ventricles of 1-day-old Sprague-Dawley rats and purified
by centrifugation on Percoll gradients. Myocytes were then cultured at
a density of 5 10
cells/cm
in a medium
containing four parts of DME (
)and one 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% CO
, the medium was changed
to one with the same composition as above, but without the serum.
Unless specified otherwise, all experiments were done at 37 °C
after 24 h of additional incubation under serum-free conditions. These
cultures contained more than 95% myocytes as assessed by
immunofluorescence staining with a myosin heavy chain
antibody(11) . For experiments on myocyte growth (Table 1) culture media also contained 0.1 mM bromodeoxyuridine during the first 48 h. The great majority of the
serum-starved myocytes were quiescent or contracted
infrequently(13) . Treatment with ouabain increased the number
of myocytes beating regularly. No attempt was made to quantitate these
effects on beating or on the contractile force. For some experiments
with myocytes, the nominally Ca
-free DME/F-12 base
medium (Sigma) was used. Incubations of myocytes in this medium for the
indicated short durations did not affect myocyte viability. Rat2 cells
(CRL 1764, American Type Culture Collection) and HeLa cells were
cultured in DME medium supplemented with 10% fetal bovine serum. The
cells (about 90% confluence) were serum-starved for 24 h before use in
experiments.
For Northern blot analysis, total RNA was isolated
using TRI reagent (Molecular Research Center, Inc.) as recommended by
the manufacturer. Routinely, 10-15 µg of total RNA was
subjected to gel electrophoresis, transferred to a Nytran membrane,
UV-immobilized, and hybridized to P-labeled probes. The
probes used for c-fos, c-jun, and GAPDH were prepared
as described before(11) . Autoradiograms obtained at -70
°C were scanned with a Bio-Rad densitometer. Multiple exposures
were analyzed to ensure that the signals were 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.
For nuclear run-on assays, myocyte nuclei were isolated as
described by Lee et al. (14) and counted in 0.04%
trypan blue using a hemocytometer. To label the nascent RNA
transcripts, the nuclei (3 10
) were incubated in
0.14 M KCl, 10 mM MgCl
, 1 mM MnCl
, 14 mM 2-mercaptoethanol, 20% glycerol,
0.2 M Tris (pH 8.0), 0.1 mg/ml creatine kinase, 10 mM phosphocreatine, 1 mM each of ATP, GTP, CTP, 0.03
µM UTP, and 100 µCi of
[
-
P]UTP (DuPont NEN, 3,000 Ci/mmol) for 15
min at 30 °C. The nuclei were collected, lysed, and digested with
RNase-free DNase (Worthington). Fifty µg of carrier yeast tRNA were
added, and the
P-labeled run-on RNA produced was then
isolated using TRI reagent (Molecular Research Center, Inc.). Purified
[
P]RNA was counted as an index of total
transcriptional activity. Equal counts of
P-labeled RNA
from different groups were used for hybridization. The probes were
applied to Nytran membrane through dot-blot apparatus, denatured, and
immobilized by UV-cross-linking.
Electrophoretic mobility shift
assay for AP-1 binding activity was done as described
previously(11) . In brief, after myocyte nuclei were isolated,
they were extracted in a solution containing 1.5 mM MgCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 26%
glycerol (v/v), 5 mM Hepes (pH 7.9), and 0.3 M NaCl.
Nuclear protein (3-5 µg) was used in the binding reaction;
the reaction mixture (20 µl) contained 50 mM NaCl, 1
mM EDTA, 1 µg of acetylated bovine serum albumin, 2 µg
of poly(dI-dC), 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10% glycerol (v/v), 5 mM MgCl
, 10 mM Tris-HCl (pH 7.5), and about
30,000 cpm of
P-labeled AP-1 probe. Control reaction
mixtures for the detection of specific binding contained 100-fold
excess of unlabeled probe. DNA-protein complexes were separated on
nondenaturing 4.5% polyacrylamide gels in 90 mM Tris borate, 2
mM EDTA (pH 8.0). Gels were vacuum-dried and exposed to x-ray
film at -70 °C for 12 to 24 h. The oligonucleotide probe for
AP-1 was synthesized according to the sequences as described by Meyer et al. (15) and labeled by T4 DNA kinase using
[
-
P]ATP.
Intracellular Ca concentration was determined using the
Ca
-sensitive fluorescent probe fura-2 as we have
previously described(16) . Briefly, the myocytes were cultured
on glass coverslips and loaded with 5 µM fura-2/AM for 30
min at room temperature. The coverslip was affixed to a culture chamber
and perfused for 15 min with the medium containing the indicated
ouabain concentration before measurements were made on about 20 single
cells. A single cell was isolated from the surrounding area using a
pinhole aperture under Nikon microscope, and single cell fluorescence
was recorded at an emission wavelength of 505 nm and excitation
wavelengths of 340/380 nm using Spex Fluoro II dual beam
spectrofluorometer (Spex Industries, Inc.). Measurement of
time-averaged signals on each single cell was completed in 30 s. The
relationship between the fluorescence ratio (340/380) and steady state
Ca
concentration was established by calibration
procedures and calculations described before (16, 23) . Because of the well-known uncertainties
associated with such calibrations, however, the indicated values should
be considered as relative changes in intracellular Ca
concentrations.
For the assay of Rb
uptake, myocytes cultured in 12-well plates were washed and
incubated in 1.0 ml of uptake medium containing 150 mM NaCl, 5
mM RbCl, 10 mM Hepes (pH 7.0), 5 mM MgCl
, 0.5 mM CaCl
, and 10 mM glucose. After 10 min of incubation with different concentrations
of ouabain,
Rb
uptake was started by the
addition of 1 µCi of
Rb
, and the
reaction was stopped after 10 min by washing four times with 3 ml of
ice-cold 0.1 M MgCl
. The cells were then treated
with 10% trichloroacetic acid at 4 °C for 60 min, and
trichloroacetic acid-soluble
Rb
was
counted. Trichloroacetic acid-precipitated cellular protein was
dissolved in 0.1 N NaOH, and protein content was
determined(17) , using bovine serum albumin as standard. DNA
was assayed fluorometrically (48) using calf thymus DNA as
standard. Myocyte viability was assayed by the extent of release of LDH
into the medium(11) .
Figure 1: Time courses of the effects of different ouabain concentrations on myocyte viability. Myocytes were prepared and incubated at 37 °C in the presence of indicated ouabain concentrations as described under ``Experimental Procedures.'' At indicated times, loss of viability was measured by the assay of released lactic dehydrogenase. Values are mean ± S.E. of 5 different experiments. S.E. not shown when smaller than symbol size.
Inhibitory effects of different ouabain
concentrations on Rb
uptake, and on
intracellular Ca
concentrations, were determined in
experiments shown in Fig. 2. Our findings on inhibition of
Rb
uptake were in general agreement with
previous observations on dose-dependent inhibitory effects of ouabain
on pump fluxes in rat neonatal cardiac myocytes (18, 19) and on
Na
/K
-ATPase activities of these
myocytes and the rat heart(2, 20) .
Figure 2:
Effects of different ouabain
concentrations on Rb
uptake by myocytes
and on intracellular Ca
concentrations. Rb
uptake was assayed as described under ``Experimental
Procedures.'' The values are means ± S.E. of 6 different
experiments. Assays of intracellular Ca
were done in
separate experiments as described under ``Experimental
Procedures.'' Values are means ± S.E. of 40-60 single
cell determinations at each ouabain concentration, using 4 different
myocyte preparations.
As expected
from a large body of previous work relating ouabain-induced inhibition
of the cardiac sodium pump to intracellular Ca(2, 3, 4, 21, 22, 23) ,
our data showed significant increases in intracellular Ca
concentrations of these myocytes in parallel with ouabain-induced
inhibition of
Rb
uptake (Fig. 2).
The following aspects of the data of Fig. 1and Fig. 2are worthy of note at this point. 1) That complete
inhibition of pump-mediated Rb
uptake
requires 1 mM or higher concentrations of ouabain (Fig. 2) is consistent with the fact that of the two isoforms
(
and
) of
Na
/K
-ATPase present in neonatal rat
cardiac myocytes, the predominant one is the relatively ouabain
insensitive
isoform(24, 25) . 2) The
observation that 0.1 mM ouabain causes significant inhibition
of the pump (Fig. 2) without affecting myocyte viability (Fig. 1) is consistent with the well-established fact that the
heart tolerates up to about 50% inhibition of
Na
/K
-ATPase without overt
toxicity(3) .
Figure 3: Dose-response curve of ouabain effects on c-fos and c-jun expressions in myocytes. A, a representative autoradiogram of ouabain effects. Serum-starved myocytes were incubated at 37 °C for 45 min with the following ouabain concentrations (µM): lane 1, 0; lane 2, 0.5; lane 3, 5; lane 4, 50; lane 5, 100; lane 6, 500. Total RNA was isolated and subjected to Northern blot analysis as described under ``Experimental Procedures.'' B, combined data from several different experiments. c-fos and c-jun mRNAs were normalized to those of corresponding GAPDH measured on the same blot and expressed relative to a control value of one.
Figure 4: Time course of ouabain-induced c-fos expression. Myocytes were treated with 100 µM ouabain and assayed for c-fos mRNA as indicated in Fig. 3after the following incubation times: lane 1, 0; lane 2, 15 min; lane 3, 45 min; lane 4, 90 min; lane 5, 3 h; lane 6, 6 h; lane 7, 18 h; lane 8, 24 h.
It was of interest to know if the above ouabain effects on serum-starved myocytes were also observed in the presence of serum. Comparison of ouabain effects on c-fos mRNA in cells cultured for 48 h in the presence of serum, with the effects in our standard myocyte preparation (24 h in serum-containing medium, followed by 24 h in serum-free medium) showed nearly identical results (Fig. 5). When serum-starved cells were exposed for 45 min to 0.1 mM ouabain, or 10% fetal bovine serum, or the combination of the two, ouabain-induced increase in c-fos mRNA was greater than that caused by serum, and the combined effects of the two agents were not greater than that of ouabain alone (data not shown). All remaining experiments were done with the serum-starved myocytes to minimize contamination by other cell types (see ``Experimental Procedures'').
Figure 5: Comparison of ouabain effects on myocyte c-fos expression in serum-free and serum-containing media. Ouabain effects were determined either on serum-starved myocytes as in Fig. 3or on myocytes similarly cultured in the presence of serum. Values are mean ± S.E. of 3 determinations.
The nuclear run-on experiments of Fig. 6showed that ouabain-induced increase in steady state concentrations of c-fos mRNA was due, at least in part, to increase in transcriptional rate of c-fos.
Figure 6:
Nuclear run-on experiments showing ouabain
effects on c-fos transcription rate. Incubations were done in
the presence of 0.5 mM ouabain and in its absence for 45 min
as in Fig. 3. Nuclei were then isolated from myocytes, labeled
with [-
P]UTP, and hybridized with
c-fos, GAPDH, and pKs (pUC19) probes. Inset, a
representative autoradiogram: lane 1, control myocytes; lane 2, ouabain-treated myocytes. Combined data (mean ±
S.E.) from 4 different experiments are shown in the graph. The
intensities of the signals of c-fos and GAPDH were corrected
by subtracting signals of pKs, and the values of c-fos signals
were normalized to those of corresponding GAPDH
signals.
To determine if the above inductions were accompanied by the formation of AP-1, containing Fos and Jun and capable of binding to DNA, nuclear extracts of control and ouabain-exposed myocytes were subjected to mobility shift assays. The results (Fig. 7) showed that ouabain increased a nuclear protein that could bind to a probe containing the AP-1 binding site. In experiments the results of which are not shown, we also noted that the nuclei of myocytes treated with ouabain contained significantly more Fos and Jun proteins than control nuclei, as determined by immunostaining procedures that we have used before to show the inductions of these proteins by other stimuli (11) .
Figure 7:
Activation of AP-1 by ouabain in myocytes.
Nuclear extracts were prepared from myocytes after treatment with 0.1
mM ouabain for 2 h, incubated with P-labeled
oligonucleotide encompassing the AP-1 motif, and subjected to mobility
shift assays. Lanes 1 and 2, control; lanes 3 and 4, ouabain-treated. Lanes 1 and 3,
P-labeled probe; lanes 2 and 4, labeled
probe plus 100-fold excess of unlabeled
probe.
Figure 8: Effects of BAPTA-AM preloading of myocytes on inductions of c-fos by ouabain and PMA. Control cells and cells that were pretreated with 10 µM BAPTA-AM for 30 min were exposed to 0.5 mM ouabain or 0.1 µM PMA for 45 min. c-fos mRNA was measured and normalized to those of GAPDH. The values are mean ± S.E. of 5 experiments.
Figure 9:
Effect of extracellular Ca on ouabain-induced increase in c-fos mRNA. Serum-starved
myocytes were placed in a nominally Ca
,
Mg
-free medium (see ``Experimental
Procedures'') supplemented with 1.2 mM Mg
and 0.1 mM EGTA. Effects of 0.5 mM ouabain, 0.1
mM phenylephrine, and 0.1 µM PMA after 45 min of
incubation in this Ca
-free medium were determined.
Ouabain effects were also determined after similar incubation in the
same medium to which 1.8 mM Ca
was also
added. Values are means ± S.E. of 3
experiments.
Preincubation of myocytes with PMA for 24 h, which is known to down-regulate PKC in these cells(27, 28) , suppressed ouabain-induced c-fos expression (Fig. 10), indicating an important role of one or more isoforms of PKC in the pathway of response to ouabain.
Figure 10: Effect of pretreatment with PMA on ouabain-induced expression of c-fos mRNA. Control myocytes and those treated with 0.1 µM PMA for 24 h were exposed to 0.1 mM or 0.5 mM ouabain for 45 min and assayed for c-fos mRNA as in Fig. 3. Values are mean ± S.E. of 6 experiments.
Figure 11: Effect of ouabain on total transcriptional activity of myocytes. Cells were incubated in the presence of 0.1 mM ouabain and in its absence for 24 h. Nuclei were prepared and subjected to run-on transcriptional analysis as described under ``Experimental Procedures.'' Values are mean ± S.E. of 5 experiments (p < 0.05).
The data presented here show that in neonatal rat cardiac
myocytes: (a) the proto-oncogenes c-fos and c-jun are rapidly induced by ouabain concentrations that inhibit the
sodium pump partially and raise intracellular Ca, but
do not cause loss of viability, (b) the sustained induction of
c-fos is dependent on extracellular Ca
and
its net influx is due to ouabain-induced inhibition of the pump, (c) PKC is likely to be involved in this pathway of
ouabain-induced gene regulation, and (d) along with the above
effects, ouabain stimulates the growth of these myocytes.
An important question is
whether the rise in intracellular Ca that is the
well-established result of the partial inhibition of cardiac
Na
/K
-ATPase (2, 3, 4) can account for the observed
induction of c-fos by ouabain. The c-fos promoter
contains two well-characterized elements, SRE and Ca
response element/cAMP response element, one or both of which are
known to be regulated by rise in intracellular Ca
in
various cells including neonatal rat cardiac
myocytes(33, 38, 39) . Since induction of
c-fos by ouabain involves PKC (Fig. 10), and SRE is
known to be stimulated by PKC(38, 40) , it seems that
ouabain induction of c-fos may be, at least in part, through
SRE. However, because of the complex and multiple mechanisms of
Ca
effects on the activations of the above two
elements of the c-fos gene(38, 39, 41) , our present data are
insufficient either to establish the definite involvement of SRE or to
rule out the role of Ca
response element/cAMP
response element in c-fos induction by ouabain. Studies aimed
at clarification of these issues are in progress.
There is
uncertainty regarding the mechanism of PKC activation in the course of
induction of c-fos by ouabain. Although increased
intracellular Ca may cause direct activation or
redistribution of PKC isoforms(28, 42) , it is not
clear that ouabain-induced increase in myocyte Ca
is
responsible for PKC activation. Previous studies have demonstrated that
both inotropic and toxic concentrations of ouabain enhance
phosphoinositide turnover, increase diacylglycerol content, and
activate PKC in the myocardium (43, 44) . While these
effects may be the consequence of activation of phospholipase C by
ouabain-induced increases in intracellular
Ca
(43) , there is also some evidence to
suggest that activation of PKC by ouabain may occur by unidentified
mechanisms that are independent of a ouabain-induced rise in
intracellular Ca
(44) .
Based on the above
considerations, it is reasonable to conclude that the same signal
(increased net influx of Ca) that accounts for the
classical effect of ouabain on contractility is also responsible for
the initiation of ouabain effect on c-fos expression, but that
additional pathways independent of the rise in intracellular
Ca
may also be involved in response of the c-fos to ouabain.
Extensive studies on hypertrophic growth of cardiac myocytes have shown that contraction stimulates growth(48, 49) , but that for some stimuli induction of growth is independent of the enhancement of contractility (30, 49) . It is also well-established that cardiac hypertrophy caused by many stimuli is associated with the selective induction of the fetal gene program(29, 30) . In these contexts, we do not have sufficient data to compare ouabain with other hypertrophic stimuli. In our experiments, ouabain clearly affected myocyte contractility as expected (see ``Experimental Procedures''), although we did not quantitate these effects. It remains to be seen if ouabain's effect on growth can be distinguished from its effects on contractility. Studies are also in progress to determine whether or not ouabain exerts selective effects on the cardiac fetal gene program.