Chronic Elevation of Calmodulin in the Ventricles of Transgenic Mice Increases the Autonomous Activity of Calmodulin-Dependent Protein Kinase II, Which Regulates Atrial Natriuretic Factor Gene Expression
Josep M. Colomer and
Anthony R. Means
Department of Pharmacology and Cancer Biology Duke University
Medical Center Durham, North Carolina 27710
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ABSTRACT
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Although isoforms of
Ca2+/calmodulin-dependent protein kinase II
(CaMKII) have been implicated in the regulation of gene expression in
cultured cells, this issue has yet to be addressed in vivo.
We report that the overexpression of calmodulin in ventricular myocytes
of transgenic mice results in an increase in the
Ca2+/calmodulin-independent activity of
endogenous CaMKII. The calmodulin transgene is regulated by a 500-bp
fragment of the atrial natriuretic factor (ANF) gene promoter which,
based on cell transfection studies, is itself known to be
regulated by CaMKII. The increased autonomous activity of CaMKII
maintains the activity of the transgene and establishes a positive
feedforward loop, which also extends the temporal expression
of the endogenous ANF promoter in ventricular myocytes. Both the
increased activity of CaMKII and transcriptional activation of ANF are
highly selective responses to the chronic overexpression of calmodulin.
These results indicate that CaMKII can regulate gene expression
in vivo and suggest that this enzyme may represent the
Ca2+-dependent target responsible for
reactivation of the ANF gene during ventricular hypertrophy.
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INTRODUCTION
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Ca2+/calmodulin-dependent protein kinase II
(CaMKII) is a multisubunit enzyme with a broad substrate
specificity. Originally isolated from mammalian brain, CaMKII is
encoded by four genes, and isoforms of the enzyme are expressed in all
vertebrate cells (1). Evidence suggests that CaMKII may be capable of
decoding the frequency of repetitive increases in intracellular
Ca2+ (2). Ca2+/CaM binding
to a subunit of a CaMKII holoenzyme complex relieves an intrasteric
autoinhibition and activates the protein kinase (1). When
Ca2+/CaM binds two adjacent subunits, one active
molecule phosphorylates the other on a single threonine residue
(T286 in CaMK
) (1). This phosphorylation has
two important consequences. First, it markedly increases the affinity
of the subunit for Ca2+/CaM, a phenomena called
CaM trapping (3). Second, the kinase activity of the subunit is
maintained even after the Ca2+ levels have
declined (4, 5, 6). The latter property has been called "autonomous"
or Ca2+/CaM-independent activity and endows
CaMKII with a memory function (5).
Whereas all of the variants of the CaMKII holoenzyme are assembled in
the cytoplasm, specific isoforms of the
,
, and
types contain
nuclear localization sequences and are targeted to the nucleus (7).
However, although the membrane and cytoskeletal roles of CaMKII are
beginning to be clarified (1), little is known about the nuclear
functions of this enzyme. Many of the studies that have addressed
nuclear functions of CaMKII have relied on the ability of C terminally
truncated, Ca2+/CaM-independent forms of the
enzyme to alter transcription when overexpressed in cells along with
selected reporter gene constructs. Such studies implicated CaMKII in
the phosphorylation of several transcription factors including CREB
(cAMP response element binding protein) (8), ATF-1 (activating
transcription factor 1) (9), CAAT-enhancer binding protein ß
(C/EBPß) (10), and SRF (serum response factor) (11). However,
the only direct experimental evidence demonstrating a requirement for
nuclear localization of a CaMKII holoenzyme in transcriptional
regulation is by Ramirez et al. (12). These authors showed
that overexpression of full-length CaMKII
B
activated transcription of the atrial natriuretic factor (ANF) gene in
cultured neonatal rat ventricular myocytes in response to an
-adrenergic agonist that increased intracellular
Ca2+. The transcriptional response could be
inhibited by KN-93, an antagonist of the multifunctional CaM kinases
(which include CaMKII), or by blocking nuclear entry of CaMKII.
Gruver et al. (13) generated several lines of transgenic
mice that specifically overexpress CaM in the heart. Transcription of
the CaM transgene was regulated by the proximal 500-bp fragment of the
ANF promoter. Analysis of two lines of mice revealed that whereas the
transgene expression was constitutive in atrial myocytes, transcription
was terminated soon after birth in ventricular myocytes (13), which is
also the fate of the endogenous ANF gene in these cells (14). CaMKII
has a much higher KCaM than many other
Ca2+/CaM-dependent enzymes (3). We reasoned that
if we could identify a line of mice that overexpressed a sufficiently
high level of CaM, the CaM might increase the autonomous activity of
CaMKII
B as this is the isoform of CaMKII
expressed in ventricular myocytes (15). In turn, if the ANF promoter
was regulated by CaMKII in vivo, the increased autonomous
CaMKII might exert a positive feed-forward effect on the ANF promoter
that controlled expression of the CaM transgene and prevent it from
being inactivated on schedule. We report that our hypothesis seems
valid and, in addition, the autonomous activity of the endogenous
CaMKII was sufficient to extend the duration of expression of the
endogenous ANF gene in ventricular cells. We propose that CaM and
nuclear CaMKII
B may play a role in the
reexpression of the ANF gene during cardiac hypertrophy.
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RESULTS
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Expression of the CaM Transgene in Ventricles of 2295 Mice as a
Function of Age
Gruver et al. (13) used DNA constructs regulated by the
proximal 500 bp of the human ANF promoter to create eight lines of
transgenic mice that specifically overexpressed proteins in the heart.
These mice produced either CaM or a mutant form of CaM called CaM-8
that bound Ca2+ normally but neither interacted
with nor activated CaM-dependent enzymes due to removal of eight amino
acids from the central helix (16). In the four lines of CaM and CaM-8
mice studied by Gruver et al. (13), the ANF transgene was
found to be regulated similarly to the endogenous ANF gene. That is,
the transgene was constitutively expressed in the atria but transiently
expressed in the ventricles as a function of age. Whereas both
transgenes and the endogenous ANF gene were expressed in ventricular
myocytes during the first postnatal week, expression was extinguished
by 21 days of age. Because we were interested in determining the
consequences of higher levels of CaM on events in the ventricles, we
examined the level of CaM expression in the hearts of newborn mice from
the remaining four uncharacterized strains. Whereas the CaM-8 transgene
behaved similarly as a function of age regardless of considerable
differences in expression at birth, three lines of CaM-overproducing
mice, including the 2295 line, exhibited a higher level of CaM at birth
and maintained this level for at least 2 weeks postnatally. Southern
and quantitative dot blot analysis revealed that the 2295 line
expressing CaM and the 4466 line expressing CaM-8 had a similar site of
transgene integration and copy number (
10 copies). Therefore, these
lines were used in our study. Figure 1
shows quantification of ventricular CaM in the 2295 line, the 4466 line
that expresses CaM-8, and in normal mice as a function of age. These
data were collected using a RIA with an antibody that equally
recognizes the CaM and CaM-8 proteins. At birth the 2295 and 4466 lines
produced 6 times more immunoreactive protein that did nontransgenic
mice of the same age. However, by day 4, the levels of immunoreactive
protein in the 4466 strain and normal mice were equivalent whereas the
2295 mice continued to demonstrate a 6-fold increase in CaM. Moreover,
this increased CaM was maintained for at least 14 postnatal days before
beginning to decline. Thus, the 2295 line afforded us the opportunity
to evaluate the mechanism responsible for maintaining the high level of
CaM past the time at which the ANF-driven transgene and endogenous ANF
gene should be silenced in ventricular myocytes. All subsequent studies
were carried out in ventricles isolated from the hearts of 14-day-old
mice.
Temporal Elevation of Ventricular CaM Levels Correlates with
Increased Autonomous Activity of CaMKII
Our hypothesis proposed that the chronically elevated levels of
CaM might result in an increase in the autonomous activity of CaMKII
that, in turn, would maintain the activity of the ANF promoter, thus
creating a positive feedback loop. As shown in Table 1
, the total activity
(Ca2+ dependent plus Ca2+
independent) of CaMKII did not differ in ventricular extracts prepared
from normal, 2295, or 4466 mice at 14 days of age. However, there was a
statistically significant increase in the autonomous CaMKII activity
(measured in the presence of EGTA) in ventricular extracts of mice
overexpressing CaM compared with those prepared from the other two
strains of mice. The protein kinase activity was quantified by
phosphorylation of the highly selective peptide substrate,
autocamtide-II in vitro (17). The isoform of CaMKII
expressed in ventricular myocytes is CaMKII
B
(15, 18). Autonomy is the result of intersubunit phosphorylation of
threonine 287 (T287) in the holoenzyme. To ensure
that the protein kinase activity in vitro reflected an
increase in the autonomous activity of CaMKII in vivo, we
used an antibody to CaMKII that only recognizes the protein when
phosphorylated on T287 (19).
T287 in
B is equivalent
to T286 in the
-isoform, and the peptide used
to raise this antibody is conserved 100% in
B
(19). As shown in the top panel of Fig. 2A
, there is a significant increase in
the amount of this immunoreactive epitope in ventricular extracts of
2295 mice relative to normal or 4466 mice. The lower panel
of Fig. 2A
shows results obtained using an antibody that recognizes
CaMKII, whether or not it was phosphorylated, and confirms that the
total amount of CaMKII does not significantly differ between extracts
from the three types of mice. Thus, both protein kinase assays and
immunoblots revealed a 2-fold increase in the autonomous activity of
CaMKII in mice that overexpress CaM in the ventricles but not change in
the total amount of CaMKII.
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Table 1. Effects of overexpression of CaM on CaN and
CaMKII in vitro activity and cAMP steady-state levels in the
ventricles of 14-day-old mice
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Figure 2. Western Blot Analysis of CaMKII and NFAT
Phosphorylation in Ventricles of 14-Day-Old Mice Overexpressing CaM
Representative Western blots of three different experiments, with at
least three normal and three transgenic mouse ventricular extracts were
performed. A, Analysis of CaMKII phosphorylated at T287.
Extracts from the indicated mouse ventricles were resolved by SDS-PAGE
and probed with an antibody specific for T287
autophosphorylated CaMKII (upper panel). The membrane
was stripped and reprobed with an antibody that recognizes CaMKII
regardless of the degree of phosphorylation (lower
panel). B, Analysis of NFAT mobility. Ventricular extracts were
resolved by SDS-PAGE as before and probed with an anti-NFAT antibody
(upper panel). The membrane was stripped and reprobed
with anti- -actinin, a myocyte-specific marker, to check for lane
loading (lower panel).
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As the expression of a transgene shows considerable variability from
mouse to mouse, we questioned whether the level of CaM was predictive
of the degree of autonomous CaMKII activity in the ventricles of
individual mice. Figure 3
is a plot of
autonomous CaMKII activity as a function of CaM levels. The calculated
correlation coefficient was r = 0.94, demonstrating that the
extent of CaMKII autonomous activity could be predicted from the total
CaM content in the ventricle with almost 90% accuracy
(r2 = 0.884).

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Figure 3. Positive Correlation between Autonomous CaMKII
Activity and CaM Levels in the Ventricles of 14-Day-Old Mice
CaM levels were measured in ventricular extracts by RIA. The same
extracts were used to measure autonomous CaMKII activity in the
presence of EGTA using autocamtide-II as the substrate. Correlation
coefficient r = 0.94; n = 18 mice.
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Like all cells, the ventricular myocyte contains a number of
CaM-dependent enzymes. We wanted to determine whether the elevated CaM
level was selectively correlated with an increase in autonomous CaMKII
activity or was more globally correlated with changes in the activity
of other CaM-dependent systems. Since the cross-sectional diameter, the
most accurate prognosticator of cardiomyocyte hypertrophy (20), is
increased in the ventricular myocytes of mice overexpressing CaM
(16.93 ± 0.83 µm compared with 11.96 ± 0.89 µm and
12.80 ± 0.15 µm in 2295, normal, and 4466 strains,
respectively, P
0.002), we evaluated two other
CaM-dependent signaling pathways that have been linked to hypertrophy.
The first is chronic ß-adrenergic stimulation (21). Although the
mechanism by which this stimulus induces hypertrophy is not known, the
ß-adrenergic receptor is coupled to Gs and thus increases the level
of cAMP. The second is the pathway by which the CaM-dependent protein
phosphatase 2B (calcineurin) controls the nuclear localization of the
transcription factor, nuclear factor of activated T cells (NF-AT), in
the cardiomyocyte (22).
The cardiomyocyte contains both a CaMK-regulated isoform of adenylyl
cyclase (type III) (23) and a CaM-dependent isoform of cyclic
nucleotide phosphodiesterase (24). Since neither of these enzymes
exhibit autonomy but the steady-state levels of cAMP reflect the
balance of the two enzyme activities, we measured the cAMP levels by
RIA. Table 1
shows that there were no differences in cAMP levels in
ventricular extracts prepared from 2295 ventricles compared with those
of normal mice.
Activation of calcineurin in ventricular myocytes has been shown to
result in dephosphorylation and nuclear translocation of NF-AT (22).
Table 1
shows that overexpression of CaM does not increase the specific
activity of calcineurin, which is not surprising as calcineurin also
does not show autonomous activity. The levels of calcineurin are not
changed in CaM-overexpressing ventricles as assessed by Western blot
analysis (data not shown) and total calcineurin activity in
vitro (Table 1
). Therefore, we examined the phosphorylation status
of NF-AT using an antibody that recognizes both phosphorylated (the
more intensely stained upper band) and dephosphorylated (the
more faintly stained lower band) forms of this protein (Fig. 2B
). The ratio of the two immunoreactive bands is not changed in
ventricular extracts prepared from normal and 2295 mice, revealing that
overexpression of CaM does not result in a change of the
phosphorylation state of NF-AT. Taken together, our results suggest
that the overexpression of CaM in ventricular cardiomyocytes
selectively increased the autonomous activity of CaMKII relative to
changes in the activity of other
Ca2+/calmodulin-dependent enzymes implicated in
pathways that lead to cardiac hypertrophy.
The Inactivation of ANF Promoter-Dependent Gene Expression Is
Delayed in Ventricles of Mice Overexpressing CaM
The fact that increased CaM levels occur in the ventricles of
14-day-old mice of the 2295 strain suggested that the ANF
promoter-driven CaM (cCaM) transgene would also remain active at this
stage. To test this possibility, we examined the level of the mRNA
expressed from the transgene by Northern blot analysis using a probe
specific for the 3'-untranslated region of chicken CaM mRNA. As shown
in the top panel of Fig. 4
, cCaM mRNA was not detected in total RNA from normal mouse ventricles
(because no transgene is present) or in total RNA isolated from
ventricles of the 4466 mice (because transgene expression has been
suppressed in the age-dependent manner typical of the endogenous ANF
gene). However, cCaM mRNA was clearly present in total RNA isolated
from the ventricles of 2295 mice at 14 days of age. Two other
CaM-overexpressing mouse lines that express less CaM than 2295 and show
different sites of transgene integration, namely 2256 and 2280, showed
temporally extended transgene expression in the ventricles relative to
the control CaM-8 animals [8 vs. 5 days, respectively (13)
and data not shown]. These results suggest that the increased CaM
levels correspond to an increase in the autonomous activity of CaMKII
which, in turn, may exert a positive feedback on the ANF promoter
driving the cCaM transgene to maintain its activity. One corollary to
this proposed feedforward loop would be that the endogenous ANF
gene should also remain active in the ventricular myocytes from the
2295 mice. As shown in the second panel of Fig. 4
, the level
of endogenous ANF mRNA is increased in 2295 ventricles relative to
either normal or 4466 mice of the same age. This experiment was
repeated with six mice from each of the three strains, and the blots
were quantified by PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) analysis. The amount of ANF mRNA in the
2295 samples was increased about 20-fold relative to either of the
other lines of mice (P < 0.01 in either
comparison).

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Figure 4. Northern Blot Analysis of Various mRNAs in the
Ventricles of Normal, 2295, and 4466 Mice
Representative Northern blot analysis of the chicken CaM transgene
expression (cCaM), endogenous ANF, skeletal -actin (sk -actin),
vascular smooth muscle -actin (vsm -actin), ventricular myosin
light chain-2 (mlc-2v), and GAPDH in ventricles from normal, 2295, and
4466 mice, as indicated. PC, Positive controls: skeletal muscle for sk
actin, and the construct for vsm -actin for the vsm
-actin probe. Ten micrograms of total RNA were used for each lane;
six mice of each strain were analyzed for every marker. The riboprobes
and specific labeled oligonucleotides used to identify each RNA are
described in Materials and Methods.
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As detailed earlier, the ventricles of the 2295 mice are significantly
larger than those of either the control or 4466 mice at 14 days of age.
The 40% increase in cross-sectional diameter of ventricular myocytes
indicates that ventricular hypertrophy accompanies the overexpression
of CaM. The ANF gene is reexpressed in ventricles of a variety of
animal models of cardiac hypertrophy (25, 26, 27, 28, 29). However, the
reexpression of the ANF gene is not a specific event as a number of
other genes characteristic of more immature cardiomyocytes are also
reexpressed. To examine the specificity of the ANF gene response in the
2295 mice, we also quantified the mRNA encoded by three other genes
known to be up-regulated in hypertrophy (30, 31, 32, 33) in the samples of RNA
from 2295, 4466, and control mice. As can be seen in Fig. 4
, neither
skeletal muscle
-actin (sk
-actin), vascular smooth muscle
-actin (vsm
-actin), or ventricular myosin light chain-2 (MLC-2v)
mRNAs are increased in the RNA isolated from ventricles of the
14-day-old 2295 mice. Thus, the continued expression of the endogenous
ANF gene is a selective response to the overexpression of CaM. As none
of the other genes we examined have been shown to be positively
regulated by CaMKII, these negative results support our contention that
the increase in autonomous CaMKII is responsible for the continued
expression of the endogenous ANF gene.
Table 2
shows that the increase in ANF
mRNA results in a 5-fold elevation in the amount of ANF peptide present
in ventricular myocytes as well as a 2-fold increase in the
circulation. To determine whether this increase in ANF results in
physiological changes that could confound our data interpretation, we
evaluated both high arterial blood pressure (63.25 ± 6.29 mm Hg;
50.17 ± 5.39 mm Hg; 56.73 ± 3.32 mm Hg in normal, 2295, and
4466 mice, respectively; P = 0.24, not significant) and
low arterial blood pressure (26.75 ± 3.31 mm Hg; 23.67 ±
3.58 mm Hg; 22.73 ± 1.22 mm Hg in normal, 2295, and 4466 mice,
respectively; P = 0.49, not significant) (28). The
absence of a significant change in blood pressure lessens the concern
that the small increase in the circulating levels of ANF has
physiological consequences. Indeed, even transgenic mice for ANF, which
show a chronic 10-fold increase in plasma levels, have no changes in
hormones (such as PRA, norepinephrine, phenylephrine, or vasopressin)
that could stimulate the heart. Nor do they reveal changes in the
expression of the endogenous ANF gene in either the atria or ventricles
(34). Therefore, none of these parameters were evaluated in the present
study.
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Table 2. Effects of overexpression of CaM in the
heart on ANF peptide levels in the ventricles and in plasma of
14-day-old mice
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During development, ANF expression is restricted to ventricular
myocytes that are located in the endomyocardial area of the wall (35),
and it is these same cells that reexpress the ANF gene in response to
hypertrophy stimuli (36). Since the 500-bp fragment of the ANF promoter
and the endogenous ANF gene are coordinately regulated in a temporal
manner in ventricles of mice that overexpress CaM, we questioned
whether the cellular specificity of the transgene and endogenous gene
was similarly coordinated. This question was addressed by in
situ hybridization using riboprobes specific for cCaM or ANF mRNA.
As shown in Fig. 5B
, at 14 days of age,
ANF expression is restricted to myocytes located in the endomyocardium
of the ventricular walls of the hearts from the 2295 strain that
overexpress CaM. However, the transgene is expressed in all ventricular
myocytes (Fig. 5E
). Thus, whereas all myocytes that express the
endogenous ANF gene also express the transgene, the overexpression of
CaM does not induce ANF gene expression in cells that do not normally
express the gene. These results also suggest that although the 500-bp
fragment of the ANF promoter is sufficient to target
cardiomyocyte-specific expression of a transgene, the promoter fragment
is missing sequences that restrict expression of the endogenous gene to
a subset of ventricular myocytes when CaM is overexpressed.

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Figure 5. Pattern of Endogenous ANF and CaM Transgene
Expression in the Heart
In situ hybridization was performed on cryosections of
hearts from normal (A, D, and G), 2295 (B, E, and H) and 4466 (C, F,
and I) 14-day-old mice (AF) or newborn mice (GI). A riboprobe
specific for the endogenous ANF (AC) or the CaM transgene (DI) was
used, and the slides were counterstained with hematoxylin and eosin, as
described in Materials and Methods. Representative
photomicrographs at low magnification are shown. Three mice from each
strain were analyzed.
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The CaM transgene mRNA is expressed in all ventricular myocytes of
14-day-old mice of the 2295 strain (Fig. 5E
), whereas no expression of
the mutant CaM transgene, CaM-8, was observed at this age (Fig. 5F
).
This result supported our original assertion that overexpression of CaM
set up a positive feedback loop that kept the transgene active whereas
CaM-8, because it did not lead to increased autonomous activity of
CaMKII, was incapable of establishing this loop. This conclusion would
be strengthened if expression of the CaM-8 transgene were restricted to
those cells that express the endogenous ANF gene, because it would show
that a CaM pathway is required for the area-extended expression. To
test this possibility, we carried out in situ hybridization
analysis on hearts of newborn mice. As shown in Fig. 5H
, the CaM
transgene is expressed in all ventricular myocytes, whereas expression
of the CaM-8 transgene is restricted to cells of the ventricular
endomyocardium (Fig. 5I
), and the pattern is very similar to that of
the endogenous ANF gene (Fig. 5B
).
Because of the transgene expression variability, we predicted that the
ventricles with initially higher CaM levels would exhibit enhanced
expression of the ANF-CaM transgene and the endogenous ANF gene
relative to the ones with initially lower levels of CaM because of the
positive feedback effect of CaM on the ANF promoter. To test this
prediction, we isolated RNA from the ventricles of 14-day-old mice of
the 2295 strain, performed Northern analysis using riboprobes specific
for the cCaM mRNA or the ANF mRNA, and quantified the amount of RNA
with a PhosphorImager. Figure 6
shows a
plot of the level of ANF mRNA as a function of the level of cCaM mRNA.
The correlation coefficient is r = 0.728, indicating a good
correlation between the expression of the ANF promoter-driven cCaM
transgene and the endogenous ANF gene.

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Figure 6. Relationship between CaM Transgene and Endogenous
ANF Gene Expression in the Ventricles
ANF and CaM transgene mRNA levels in individual ventricles were
quantified from a Northern blot using a PhosphorImager. The values were
normalized to GAPDH mRNA levels and expressed as relative units.
Correlation analysis shows an r = 0.728; n = 11.
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DISCUSSION
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We report that chronic overexpression of CaM in the ventricular
myocytes leads to an increase in the degree of autophosphorylation and
autonomous activity of endogenous CaMKII in a dose-dependent manner. In
turn, the increase in CaMKII activity results in an extended temporal
expression of both the ANF promoter-driven CaM transgene and the
endogenous ANF gene. Both the activation of CaMKII and the increase in
ANF promoter-mediated gene expression are highly selective consequences
of the overexpression of CaM. These data are the first to demonstrate a
clear association between the degree of autonomous CaMKII activity and
regulation of gene expression in vivo in a mammalian
system.
Although we cannot directly determine the isoform of CaMKII responsible
for the regulation of ANF gene expression, we highly suspect that it is
the nuclear form of CaMKII
, CaMKII
B. The
CaMKII
gene is expressed in the heart, and the
B-isoform is known to contain a nuclear
localization signal (7, 15). Previously, Ramirez et al. (12)
used transient transfection to show that overexpression of CaMKII
B in cultured ventricular myocytes isolated
from neonatal rats transcriptionally regulates a cotransfected
transgene driven by a 638-bp fragment of the rat ANF promoter. The
authors showed that the CaMKII effect occurred only with CaMKII
isoforms capable of entering the nucleus and required an intact serum
response element (SRE) in the ANF promoter. Since CaMKII can
phosphorylate the SRF on Ser103 (11) and this
modification has been suggested to increase SRF-mediated transcription
(37), Ramirez et al. (12) suggested that phosphorylation of
SRF might be the mechanism by which CaMKII regulates transcription of
the ANF gene. However, we failed to demonstrate an increase in
Ser103 phosphorylation of SRF or an increase in
the amount of SRF in vivo (data not shown). Thus, it is
unlikely that regulation of SRF is the primary mechanism by which
CaMKII enhances ANF gene transcription in mouse ventricular myocytes
in vivo.
It is not clear why the ANF promoter seems to be selectively targeted
by overexpression of CaM and increased activity of CaMKII. Stimulation
of the heart by other agents that increase intracellular
Ca2+ also leads to increases in ANF gene
transcription but the cellular transcriptional response appears to be
much less selective. For example,
1-adrenergic
agonists increase intracellular Ca2+ and activate
ANF gene expression in cultured ventricular myocytes (38). However,
this stimulus also increases transcription of the skeletal
-actin
and mlc-2v promoters (32, 39). Renin, which binds to a Gq-coupled
receptor present on the myocyte membrane, activates PLC
, and
increases intracellular Ca2+ (40), also regulates
ANF and skeletal
-actin gene expression (27). These same two genes
are up-regulated by overexpression of a
Ca2+/CaM-independent form of calcineurin in the
heart (22). Interestingly, although all three genes respond to an
increase in Ca2+, the promoters contain different
regulatory elements (41, 42, 43). It seems possible that multiple
Ca2+/CaM-dependent pathways are involved in
orchestrating the overall transcriptional response to a rise in
intracellular Ca2+ and that activation of CaMKII
is only rate limiting for transcription of the ANF gene.
Initially, we questioned the selectivity of the CaMKII effect as other
CaM-dependent pathways exist in the myocyte that potentially could
alter transcriptional responses. One such pathway involves the
Ca2+/CaM-dependent protein phosphatase 2B, or
calcineurin. Activation of this enzyme has been implicated in the
development of cardiac hypertrophy because it controls the subcellular
localization of the transcription factor NFAT (22). Originally
identified in T lymphocytes, this regulatory pathway occurs in a number
of other cells including myocytes (22). Increased synthesis of
calcineurin in response to myocyte stimulation (44) results in
dephosphorylation of a subunit of NFAT, which is required for this
transcription factor to enter the nucleus (45). We did not find
calcineurin activity or levels to be increased by chronic
overexpression of CaM, nor did we detect a difference in the degree of
phosphorylation of NFAT, which can be readily observed by an altered
mobility on polyacrylamide gels (46). This might not be surprising
since calcineurin does not exhibit an autonomous state and the myocytes
were not subject to stimulation. In addition, calcineurin may be
subject to activity-induced inactivation due to oxidation, possibly of
its cofactor iron. This latter mechanism, which couples
Ca2+/CaM-dependent protein dephosphorylation to
the redox state of the cell, provides a way to reversibly desensitize
the enzyme (47).
Two other CaM-dependent kinases have been linked to regulation of gene
expression in other systems (48). However, one of these enzymes, CaM
kinase IV, is not expressed in mouse ventricles based on our in
situ hybridization studies (data not shown). The other enzyme, CaM
kinase I (CaMKI), is expressed in the heart (49), but a
Ca2+/CaM-independent form of CaMKI cannot
increase the activity of an ANF promoter-derived reporter gene in
ventricular myocytes (50). Thus, CaMKI is an unlikely mediator of ANF
gene transcription in vivo.
An alternative pathway by which an elevation of CaM could alter
transcription involves a modification of cAMP levels, which would
regulate protein kinase A (PKA). PKA is known to phosphorylate a number
of transcription factors. The heart contains
Ca2+/CaM-dependent and independent isoforms of
both cyclic nucleotide phosphodiesterase (24) and adenylyl cyclase (23, 51). However, we did not detect any increase in the steady-state levels
of cAMP, suggesting that these signaling pathways may not be
chronically modified by the overexpression of CaM. Together, the data
support our contention that the increase in autonomous activity of
CaMKII is a selective consequence of the elevation of CaM levels and
that the increase in the activity of CaMKII is responsible for the
increased transcriptional activity of the transgene and endogenous ANF
promoters.
Why would an increase in CaM lead to such a selective activation
of CaMKII? CaMKII is an unusual CaM-dependent enzyme in several
respects. First, when in its inactive state, it has one of the lowest
affinities for Ca2+/CaM of any
Ca2+-dependent CaM-binding enzyme
(Kd
45 nM) (3). Second, at the
resting concentration of Ca2+ common to many
cells (100200 nM) CaMKII does not bind CaM (3). Third,
when adjacent subunits of the holoenzyme complex bind
Ca2+/CaM, one subunit phosphorylates its
neighboring subunit on T287 (1). This
phosphorylation event increases the affinity of CaM-binding 1000-fold
and generates an enzyme that is active even when CaM becomes
dissociated (3). Thus, higher concentrations of
Ca2+ are required to activate CaMKII than for
most other CaM-dependent enzymes, but once activated, the kinase is
independent of CaM binding. Increasing the CaM concentration markedly
decreases the amount of Ca2+ required for target
enzyme activation (52). Therefore, it is possible that increasing the
CaM concentration in the myocyte allows CaMKII activation to occur at
concentrations of Ca2+ that would normally be
insufficient for its activation. Indeed, precedence exists for this
suggestion as Wang and Kelly (53) revealed that injection of CaM into
postsynaptic neurons decreased pulse-paired facilitation at ambient
Ca2+ concentrations. Once activated, CaMKII
activity becomes Ca2+/CaM autonomous and
inactivation requires dephosphorylation of T287
(1). The continued presence of elevated CaM levels would favor a
steady-state increase in autonomous CaMKII activity. Since both the
regulatory regions of the CaM transgene and the endogenous ANF gene
require the action of CaMKII, a feed-forward loop would be established
whereby the increased CaM would activate CaMKII which, in turn, drives
the transcription of the transgene.
Whereas the 500 bp fragment of the ANF promoter is sufficient to target
expression of the cCaM transgene to cardiomyocytes, it lacks the
specificity of the endogenous ANF gene and is expressed in all
ventricular cells. Thus, the increase in CaMKII activity might be
expected to also occur in all ventricular myocytes. On the other hand,
the endogenous ANF gene is only expressed in cells that comprise the
endomyocardial wall (35, 36). These are the same cells that express ANF
during early development and reexpress ANF in response to hypertrophic
stimuli. This suggests that the regulatory elements that are
responsible for the regionally restricted expression of the ANF gene
must occur upstream of the 500-bp fragment used to drive the transgene.
Nevertheless, our results suggest that CaMKII may play a role in
regulating the reexpression of the ANF gene that accompanies cardiac
hypertrophy. Support for this possibility comes from Kirchhefer
et al. (54), who reported an increased CaM kinase activity
in human hypertrophied ventricles although the specific CaM kinase was
not identified. However, the nuclear isoform of CaMKII
, CaMKII
B, has been shown by Hoch et al.
(55) to be up-regulated in human hearts that have dilated ventricles.
Our results are the first to demonstrate that increased activity of
endogenous CaMKII (presumably CaMKII
B)
correlate with increased ANF gene expression in vivo and
provide the basis for future experiments aimed at elucidating the
Ca2+-dependent signaling pathways involved in
the generation of cardiac hypertrophy.
 |
MATERIALS AND METHODS
|
---|
Experimental Animals
Mice were housed in the Duke University Levine Science Research
Center Vivarium under a 12-h light, 12-h dark cycle. Food and
water were provided ad libitum, and care was given in
compliance with NIH guidelines on the use of laboratory and
experimental animals. All transgenic mouse strains used in this study
were generated by Gruver et al. (13). Animal experiments
were carried out in compliance with a protocol approved by the Duke
University Animal Care and Use Committee.
RIAs for CaM, cAMP, and ANF
Ventricles were assayed for total soluble protein by the
Bradford procedure (Bio-Rad Laboratories, Inc., Hercules,
CA) after hearts were harvested from 14-day-old mice (unless otherwise
stated) of normal, 2295, or 4466 strains (13). The ventricles were
microdissected and homogenized twice with 1-min bursts of a Polytron
homogenizer (Brinkmann Instruments, Inc. Westbury, NY) at
a setting of 4.5 using a PT-10 generator, in RSB (10 mM
Tris pH 7.4, 30 mM NaCl, 3 mM
MgCl2) containing 10 mM
3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate,
several proteinase inhibitors (1 mM phenylmethylsulfonyl
fluoride, 1 mg/ml Pefablock, 10 µg/ml aprotinin, 20 µg/ml trypsin
inhibitor, 10 µg/ml leupeptin, and 1 µg/ml pepstatin A) and several
phosphatase inhibitors (1 µM microcystin, 100
µM Na3VO4, 20
mM NaF). After the samples were centrifuged for 10 min, the
soluble fraction was used for the indicated assays.
CaM protein levels were determined in ventricular extracts by RIA,
exactly as previously described (56).
cAMP levels in ventricular extracts were assayed using a
[8-3H]cAMP assay system (Amersham Pharmacia Biotech, Buckinghamshire, UK). After homogenization of
fresh ventricles in RSB buffer containing 10 mM CHAPS and 4
mM EDTA, the extracts were heated for 5 min in a boiling
water bath, and centrifuged for 10 min and the supernatant was assayed
exactly as indicated by the manufacturer.
ANF peptide levels were determined by RIA both in ventricular extracts
and plasma, using the [125I]
ANP RIA
(Amersham Pharmacia Biotech), which recognizes mouse ANF
with an efficiency of 95%. To assay tissue-immunoreactive ANF,
ventricles were dissected from 14-day-old mice, weighed, and quickly
frozen in liquid nitrogen. The tissue extraction was done as described
(57). Briefly, the frozen ventricles were boiled in screw cap tubes
containing 1 ml of 1 M acetic acid with 0.1 N
HCl for 5 min, homogenized with a Polytron tissue homogenizer, and
centrifuged for 30 min at 10000 g at 4 C. The supernatant was
transferred to 4 ml polypropylene tubes and 2 ml of 2 M
Tris-HCl pH 7.4 were added to neutralize. Aliquots of 100 µl were
used to assay ANF according to the manufacturers instructions. The
final ANF amounts were normalized to initial ventricular wet weight. To
assay plasma ANF, the manufacturer instructions were followed,
including the extraction of ANF with Amprep C8 columns (Amersham Pharmacia Biotech).
Enzyme Assays
Ventricular homogenates were assayed for CaMKII activity in 50
µl of a reaction mixture consisting of 50 mM HEPES pH
7.5, 10 mM MgCl2, 0.5 mM
dithiothreitol (DTT), 1 µM CaM, 100 nM
microcystin, 50 µM ATP (1500 cpm/pmol
[
-32P]ATP), and 0.1 mM of
substrate peptide autocamtide II.
Ca2+/calmodulin-dependent kinase activity was
determined by including 1 mM CaCl2 in
the reaction mixture, while autonomous activity was measured in the
presence of 2.5 mM EGTA. The reaction was carried out for 2
min at 30 C and 40-µl aliquots of the reaction mixture were spotted
onto P81 phosphocellulose filters (Whatman, Clifton, NJ)
as described previously (58).
Inhibitor-1 protein was labeled with
[
-32P]ATP, modified from Stewart et
al. (59) as follows. Glutathione-S-transferase
(GST)-inhibitor-1 fusion protein (46 mg) was incubated with a
solution containing 100 µM ATP (200 µCi/ml of
[
-32P]ATP), 1 mM
MgCl2, 50 mM HEPES, 100 U
of PKA in a final volume of 1 ml at 37 C for 5 h. Aliquots of 2
µl were taken every 30 min to determine [32P]
incorporation into GST-inhibitor-1 (2 µl of reaction were added to
600 µg of BSA and 1 ml of 25% TCA, kept on ice for 5 min,
centrifuged for 2 min, washed again, and counted). Once saturation was
achieved, [
-32P]ATP was separated from
[32P]GST-inhibitor-1 using an Amicon-30
concentrator (Amicon Inc., Beverly, MA). Five hundred microliters of
the reaction were added to 1500 µl of 500 mM
glycero-phosphate, loaded onto an Amicon-30 concentrator, and
centrifuged at 3000 rpm for 1 h. After the concentrator was washed
with 2 ml of H2O,
[32P]GST-inhibitor-1 protein was eluted by
resuspending it in 2 ml of H2O,
inverting the concentrator, and centrifuging 10 min at 1000
rpm.
A modification of the method of King et al. (60) was used to
assay calcineurin activity. Ventricular homogenates (40 µg) were
added to a reaction mixture containing a final concentration of 50
mM HEPES, pH 7.5, 5 mM
MgCl2, 0.1 mM
MnCl2, 0.5 mM DTT, 0.1
mM EDTA,
10-8
M microcystin-LR, 0.1 mM
CaM, 1.5 µM
[32P]inhibitor-1 as a substrate, and 0.2
mM CaCl2. Controls without
Ca2+ were done in the presence of 1
mM EGTA. The reaction was carried out for 10 min
at 37 C and stopped by placing the tubes on ice after adding 100 µl
of TCA 25% and 100 µl of 6 mg/ml of BSA. After centrifugation for 5
min at 4 C, 200 µl of the supernatant were counted with 2 ml of
scintillation liquid (ultrafluor).
Western Immunoblot Analysis
After homogenizing the ventricles as described before, the
ventricular soluble fraction was run on a 6% or a 10%
SDS-polyacrylamide gel for NFAT or CaMKII Western blot analysis,
respectively. The proteins were transferred to an Immobilon-P membrane
(Millipore Corp., Bedford, MA), which then was blocked for
1 h in TBST (25 mM Tris, pH 7.4, 140 mM
NaCl, 3 mM KCl, 0.1% Tween-20), containing 5% nonfat dry
milk and 0.5% cold water fish skin gelatin (Sigma) as
blocker agents. Subsequently, the filter was incubated overnight with
the indicated antibody. An anti-NFAT antibody that recognizes all the
isoforms of NFAT (sc-1149, Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA) was used at a 1:500 dilution or a monoclonal antibody
specific for CaMKII phosphorylated at T286
[22B1, a generous gift of Dr. M. K. Kennedy (19)] at a 1:1000
dilution. After 46 washes with TBST, the filter was incubated for
1 h in the same buffer with 5% nonfat dry milk and 0.5% gelatin,
containing a second antibody labeled with horseradish peroxidase
(Amersham Pharmacia Biotech, Arlington Heights, IL). Then
the membranes were washed again in TBST, incubated with enhanced
chemiluminescence (ECL) reagents (Amersham Pharmacia Biotech) as indicated by the manufacturer, and exposed to film
(Eastman Kodak Co., Rochester, NY). Afterward, membranes
were stripped (according to ECL protocol), and reprobed with another
antibody, as indicated in the figures. An anti
-actinin (A-7811,
Sigma) was used at 1:800 dilution or a polyclonal antibody
specific for CaMKII [a generous gift of Dr. M. K. Kennedy (19)]
at a 1:1000 dilution. The subsequent washes and second antibody
incubation were performed as described above.
RNA Analysis
Total RNA from microdissected ventricles was isolated using the
Ultraspec RNA kit (Biotex Laboratories, Inc., Houston, TX)
and electrophoresed through denaturing 1.5% agarose, 6% formaldehyde
gels. RNA was blotted onto Zeta probe membranes and hybridized to
riboprobes specific for chicken CaM (cCaM), rat ANF, mouse skeletal
muscle
-actin, or mouse vascular smooth muscle
-actin, as
specified, in a buffer containing 50% formamide, 1.5x SSPE, 1% SDS,
0.5% Blotto, 0.2 mg/ml yeast RNA, 0.5 mg/ml salmon sperm DNA, and
[32P]riboprobe. cRNA CaM riboprobe was
generated from the 3'-end of the chicken CaM cDNA as described
previously (13). cRNA ANF riboprobe was generated from a plasmid (pGEM)
containing a 0.6-kb PstI fragment of rat ANF (61), using SP6
RNA polymerase after plasmid linearization with HindIII
according to the Promega protocol (Promega Corp., Madison,
WI). cRNA skeletal
-actin riboprobe was generated from a Bluescript
plasmid containing 240 bp of the 3'-untranslated region of mouse
skeletal muscle
-actin (generous gift of Dr. R. J. Schwartz),
using T3 RNA polymerase. cRNA mouse smooth muscle
-actin riboprobe
was generated from Bluescript plasmid containing 160 bp
(DdeI-EcoRI fragment) of the 3'-untranslated
region of mouse vascular smooth muscle
-actin (62), using T3 RNA
polymerase. When oligonucleotides were used as probes, the membranes
were hybridized in a buffer containing 5xSSC, 20
mM sodium phosphate (pH 7.2), 7% SDS, 1x
Denhardts, 0.1 mg/ml salmon sperm DNA, and
[32P]oligoprobe. The oligonucleotide probe used
for myosin light chain-2v, which has been previously described (63),
was labeled using T4 polynucleotide kinase. When quantification was
required, the membranes were stripped (according to the Bio-Rad Laboratories, Inc. instructions) and reprobed with
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA, using a 780-bp
fragment (PstI-XbaI) of the human GADPH
cDNA containing the 5'-UT region and the sequence encoding the first
250 amino acids (64). ANF and MLC-2v mRNA levels, determined by
Northern analysis, were normalized to GAPDH mRNA levels after
quantification by a PhosphorImager.
In Situ Hybridization
Hearts were rinsed in PBS, frozen in Isopentane at -30 C, and
kept in a -80 C freezer. Sections (10 µm) were obtained with a
Frigocut cryostat (Leica Corp., Nussloch, Germany)
at -20 C and thaw-mounted on RNAse free silylated glass
microscope slides (CEL Associates Inc, Houston, TX) and stored at -80
C. Frozen slides were fixed in ice-cold 4% paraformaldehyde in PBS for
10 min, rinsed with DEPCed 2xSSC (SSC: 150 mM NaCl, 15
mM sodium citrate, pH 7.0) and the sections were
illuminated with a UV lamp for 5 min to postfix the biological
material. After the sections were incubated with the prehybridization
buffer (50% formamide, 0.6 M NaCl, 10 mM
Tris-HCl, pH 7.5, 1 mM EDTA, pH 8.0, 50 µg/ml salmon
sperm DNA, 500 µg/ml yeast total RNA, 50 µg/ml yeast transfer RNA)
in a humid oven at 50 C for 1 h, the RNA was hybridized with the
hybridization buffer (50% formamide, 0.6 M NaCl, 10
mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 8.0, 10
µg/ml salmon sperm DNA, 50 µg/ml yeast total RNA, 50 µg/ml yeast
transfer RNA, 10% dextran sulfate) containing 5000 cpm/µl of a
heat-denatured (15 min at 65 C), [35S]RNA probe
overnight at 50 C. After hybridization the slides were washed,
dehydrated, developed, and counterstained in hematoxylin-eosin as
described previously (65). cRNA CaM and cRNA ANF were obtained from the
same constructs used for Northern blots, but they were labeled
instead with rUTP, according to the Promega Corp.
protocol. The specific activity of the
[35S]riboprobes was higher than
108 cpm/µg RNA.
Statistical Analysis
ANOVA, Students t test, and regression analysis
were done using Statview (Abacus Concepts Inc., Berkeley, CA).
Statistical significance was accepted when P <
0.05.
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to Dr. R. J. Schwartz (Baylor College of
Medicine, Houston, TX) for providing us with mouse skeletal
-actin
probe and Dr. M. B. Kennedy (Caltech, Pasadena, CA) for kindly
sharing her specific antibodies for CaMKII. We also gratefully
acknowledge Dr. H. A. Rockman (Duke University) for measuring blood
presure in mice.
J. Colomer is a recipient of a Formación de Personel
Investigador fellowship from the Spanish Government. This
research was supported by NIH Grants HD-07503 and GM-33976 (to
A.R.M.).
 |
FOOTNOTES
|
---|
Address requests for reprints to: Anthony R. Means, Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710. E-mail: tony.means{at}duke.edu
Received for publication February 21, 2000.
Revision received April 12, 2000.
Accepted for publication April 13, 2000.
 |
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