(Circulation. 1997;96:3943-3953.)
© 1997 American Heart Association, Inc.
Articles |
cis-Acting Sequences That Mediate Induction of ß-Myosin Heavy Chain Gene Expression During Left Ventricular Hypertrophy due to Aortic Constriction
From the Cardiovascular Division, Departments of Medicine and Cell Biology, Albert Einstein College of Medicine, Bronx, NY (K.H., S.J.L., R.N.K.), and the Department of Cell Biology, Parke-Davis Pharmaceutical Research Division, Warner Lambert Co, 2800 Plymouth Rd, Ann Arbor, Mich (S.M.J., B.E.M.).
Correspondence to Richard N. Kitsis, Departments of Medicine (Cardiology) and Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY 10461. E-mail kitsis{at}aecom.yu.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsAnimal Surgery and Tissue...RNA AnalysisStatistical AnalysesResultsTransactivation of a ~3542...ß-MHC GATA Site Can...DiscussionReferences |
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Background Marked alterations in the expression of specific genes occur during the development of cardiac hypertrophy in vivo. Little is known, however, about the cis-acting elements that mediate these changes in response to clinically relevant hypertrophic stimuli, such as hemodynamic overload, in intact adult animals.
Methods and Results The left ventricular expression
of a directly injected reporter gene driven by 3542 bp of rat
ß-myosin heavy chain (ß-MHC) promoter was increased 3.0-fold by
aortic constriction (P<.005), an increment similar to
the 3.2-fold increase in the level of the endogenous
ß-MHC mRNA in the same left ventricles. Subsequent analysis
identified a 107-bp ß-MHC promoter sequence (-303/-197) sufficient
to convert a heterologous neutral promoter to one that is
activated by aortic constriction. These sequences contain two
M-CAT elements, which have previously been demonstrated to mediate
inducible expression during 
1-adrenergic–stimulated
hypertrophy in cultured neonatal cardiac myocytes, and a
GATA element. Although simultaneous mutation of both M-CAT
elements markedly decreased the basal transcriptional activity of an
injected 333-bp ß-MHC promoter, it had no effect on aortic
constriction-stimulated transcription (3.5-fold increase,
P<.005 for both wild type and mutant). In contrast,
mutation of the GATA motif markedly attenuated aortic
constriction-stimulated transcription (1.6-fold, P=NS)
without affecting the basal transcriptional activity. This GATA site
can interact with in vitro translated GATA-4 and compete with an
established GATA site for GATA-4 binding activity in nuclear extracts
from aortic constricted hearts.
Conclusions Basal and aortic constriction-stimulated transcription of the ß-MHC gene is mediated, at least in part, through different mechanisms. A GATA element within ß-MHC sequences -303/-197 plays a role in the transcriptional activation of this gene by aortic constriction.
Key Words: genes • hypertrophy • signal transduction
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Introduction |
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TopAbstractIntroductionMaterials and MethodsAnimal Surgery and Tissue...RNA AnalysisStatistical AnalysesResultsTransactivation of a ~3542...ß-MHC GATA Site Can...DiscussionReferences |
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Hemodynamic overload is a complex physiological stimulus, consisting of humoral, mechanical, and neural components, which elicits marked changes in myocardial structure and function. These include myocyte hypertrophy, chamber dilation, and mechanical failure. Although hemodynamic overload may arise from a wide variety of primary abnormalities, including hypertension, myocardial infarction, and valvular heart disease, its effects on the myocardium play a role in virtually all forms of heart failure. Hemodynamic overload-induced hypertrophy results in marked alterations in cardiac gene expression. These are characterized by transient induction of selected immediate early genes and more prolonged reactivation of cardiac genes whose expression in the ventricle is limited primarily to fetal life. The latter are exemplified by skeletal
-actin, ß-myosin heavy
chain (MHC), and atrial natriuretic factor
(ANF).1–6 In contrast, expression of -MHC,
the major adult isoform of myosin heavy chain in the rodent, is
down-regulated7,8 during
hemodynamic overload hypertrophy,
demonstrating that these alterations do not represent
generalized increases in transcription associated with cardiac growth.
Currently, little is known about the precise molecular mechanisms by
which hemodynamic overload transduces these changes in
gene expression during the development of cardiac
hypertrophy.
Previous work to delineate the regulation of cardiac gene expression
during myocyte hypertrophy has been performed using
transient transfections into primary cultures of neonatal rat cardiac
myocytes.9–16 Treatment of these cells with
1-adrenergic
agonists,9,11,12,14,16 various growth
factors,10,13 and mechanical
stretch15 stimulates increases in myocyte volume
and reproduces many of the changes in cardiac gene expression
characteristic of the hypertrophic program in vivo. Despite these
similarities, several features of these cell culture systems raise
questions about their ability to model adult cardiac myocytes
undergoing hemodynamic overload-induced
hypertrophy in vivo. First, myocardial development is not
complete at birth and differences in gene expression exist between
neonatal and adult cardiac myocytes (for reviews see refs. 1 to 3).
Second, differences in gene expression have been observed between
dissociated myocytes in culture and those in the intact
heart.17 Third, the time course
for the hypertrophic response in vitro is much more rapid than that
observed in vivo. Fourth, it is unclear whether a complex
physiological stimulus such as
hemodynamic overload can be modeled accurately by a
single humoral or mechanical stimulus. For example, although myocardial
activation of 1-adrenergic receptors can lead
to cardiac hypertrophy in vivo,18
there is little evidence that this mechanism plays a critical role in
disease-related hypertrophic growth. For all of these reasons, it would
be advantageous to have a system in which to study cardiac gene
regulation in the adult heart in response to a well recognized and
clinically important hypertrophic stimulus such as
hemodynamic overload.
To accomplish this, we and others have developed a means of transferring genes into striated muscle by the simple direct injection of naked plasmid DNA in vivo.19–26 Transfection by this approach occurs specifically in myocytes.19–22,24 In addition, the expression of an injected construct driven by promoter sequences from a cardiac gene is regulated in parallel with that of the endogenous cardiac gene from which the promoter sequences were derived.23,25 This approach has been used to investigate the transient induction of c-fos expression in response to intraventricular balloon inflation in isolated perfused hearts.26 In the current study, we investigated the roles of ß-MHC and ANF promoter sequences in mediating induction in the expression of these genes in the left ventricles of intact adult rats during hypertrophy evoked by aortic constriction. These genes were chosen for analysis because the accumulation of their products closely parallels the development of hemodynamic overload-stimulated cardiac hypertrophy.4,8
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsAnimal Surgery and Tissue...RNA AnalysisStatistical AnalysesResultsTransactivation of a ~3542...ß-MHC GATA Site Can...DiscussionReferences |
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Plasmids
The reporter plasmid p-3542ß-MHCluc was constructed by cloning
a BamHI-XmnI fragment encompassing nucleotides -3542 to +89
relative to the transcriptional start site of the rat ß-MHC gene
(generous gift of Dr. John G. Edwards, New England Regional Primate
Center) into BamHI-SmaI digested pXP2,27
containing the firefly luciferase (luc) coding region and SV 40
splicing and polyadenylation signals. Reporter plasmids carrying
further 5' deletions of this promoter (with the same 3' end at +89)
were generated by using HindIII (p-1145ß-MHCluc) or NheI
(p-408ß-MHCluc) sites, or by amplifying a part of ß-MHC promoter
sequences with polymerase chain reaction (PCR) (p-303ß-MHCluc and
p-203ß-MHCluc) and subcloning into a BamHI-BglII-cleaved pXP2. M-CAT
and GATA mutations were studied in the context of ß-MHC sequences
-333 to +34. p-333wtß-MHCluc (wild type) was generated by PCR.
Combined site-directed mutagenesis of the distal (-274 to -280) and
proximal (-204 to -210) M-CAT elements was performed in two steps.
First, constructs with a mutation in either distal or proximal M-CAT
element were generated by PCR. Second, a promoter with mutations in
both M-CAT elements (p-333mutA+Bß-MHCluc) was generated by inserting
the PflMI fragment of the plasmid containing the proximal M-CAT
mutation into the corresponding PflMI sites in the distal M-CAT mutant.
p-333mutGATAß-MHCluc was generated by PCR. ptkCAT contains the
chloramphenicol acetyltransferase (CAT) gene under the transcriptional
control of the herpes simplex virus thymidine kinase promoter sequences
-109 to +56.28 p-303/-197tkCAT construct was
constructed by amplifying the ß-MHC sequences from
nucleotide -303 to -197 with PCR and subcloning this
product into XbaI-digested tkCAT. p-3412ANFluc was constructed by
fusing the BglII fragment of ANF-pKC7 (generous gift of Dr. Christine
E. Seidman, Harvard),29 which includes
nucleotides -3412 to +63 of the rat ANF gene, into
BamHI-BglII-digested pXP2. pRSVCAT and pRSVluc, containing Rous sarcoma
virus (RSV) long-terminal repeat sequences spliced to CAT and luc
respectively, have been described
previously.21–23,30 All constructs cloned using
PCR were verified by sequencing in both directions. Plasmids were
purified by anion exchange chromatography (QIAGEN,
Chatsworth, CA), quantitated by measurement of
OD260, and examined on agarose gels stained with
ethidium bromide prior to use.
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Animal Surgery and Tissue Preparation |
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TopAbstractIntroductionMaterials and MethodsAnimal Surgery and Tissue...RNA AnalysisStatistical AnalysesResultsTransactivation of a ~3542...ß-MHC GATA Site Can...DiscussionReferences |
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A total of more than 400 adult female Wistar rats weighing between 175 and 200g were used for these experiments. Cardiac injection was performed as described previously.21–24 Eighty µl of a solution containing closed, circular plasmid DNA and 3% Evans blue dye (to confirm the intramuscular location of the injection) in 0.9% NaCl were injected through a 27-gauge needle. Plasmid DNA consisted of 5 µg of the reporter construct driven by the cellular promoter of interest and 0.5 µg of pRSVCAT or pRSVluc to correct for variation in transfection efficiency. These dosages were chosen based on preliminary experiments showing that the amount of luciferase activity in cardiac homogenates increases linearly following injection of 0 to 5 µg of this plasmid.24 On the day following injection, animals were subjected to either abdominal aortic constriction or sham operation. Constriction of the aorta above the suprarenal arteries was accomplished by tying a 3-0 silk suture securely around both the aorta and a short segment of a 23 gauge needle, and then removing this needle. In sham operated rats, abdominal aortae were exposed but no ligature placed. Surgical mortality was less than 5% in both sham operated and aortic constricted groups. Post-operative mortality was approximately 40% in the aortic constricted group and 1% in the sham operated group. Animals were sacrificed 12 days following aortic constriction (13 days after injection). This time point was chosen because: a) expression of directly injected genes are maximal and constant 5 to 14 days post-injection;21–24 b) induction of the endogenous ß-MHC gene in adult rodent ventricles is temporally associated with the development of cardiac hypertrophy and more marked in later stages of hemodynamic overload;1–4,8 and c) induction of ß-MHC expression is more uniform throughout the ventricle at later time points following aortic constriction compared with its earlier localization in the subendocardium and surrounding intracardiac vessels.31 Following sacrifice, hearts were removed, atria and great vessels trimmed, ventricles washed in iced-cold phosphate buffered saline, and left ventricle with interventricular septum, right ventricle, and lungs weighed. Then, the basal one-third of the left ventricle was discarded to avoid contamination with atrial tissue. The middle one-third of the left ventricle was immediately frozen in liquid nitrogen for subsequent RNA analysis, and the apical one-third (~300 mg) was homogenized in 1.5 volumes of ice-cold homogenization buffer32 with a Tissumizer (Tekmar Co., Cincinnati, Ohio). Homogenates were centrifuged at 7000g for 30 minutes at 4°C, and supernatants were used for luc and CAT analyses. In all experiments, aortic constriction was considered successful only if the resulting left ventricular/body weight ratio was
30%
higher than that of the mean of the animals subjected to sham operation
at the same time. By this criterion, < 10% of animals were excluded
from analysis. The protocol was approved by the Ethics
Committee of Animal Institute of the Albert Einstein College of
Medicine.
Reporter Gene Assays
Both luc and CAT activities were assayed in the same supernatant
of cardiac homogenates as described
previously.21–24 Using a Monolight luminometer
(Model 2010, Analytical Luminescence Laboratory, San Diego, Calif.),
luc activity was measured in 15 µL aliquots of the supernatant. For
CAT activity, 15 µL of the supernatant were assayed with an
incubation time of 2 hours for samples in which 0.5 µg pRSVCAT was
injected. For those in which 5 µg of ptkCAT was injected, 100 µL of
the supernatant was assayed with an incubation time of 12 hours.
Samples in which CAT conversion was less than 0.5% (background
averaged 0.1%) were not reported. By this criterion, no more than two
animals in any group were excluded, and no groups were selectively
affected. CAT conversions were within the linear range for all
determinations reported in this paper. Results are expressed as
luc-background (in raw luminometer units)/CAT-background (as
percent conversion) in p-ß-MHCluc/p-RSVCAT and p-ANFluc/p-RSVCAT
injections and as CAT-background/luc-background in p-tkCAT/p-RSVluc
injections.
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RNA Analysis |
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TopAbstractIntroductionMaterials and MethodsAnimal Surgery and Tissue...RNA AnalysisStatistical AnalysesResultsTransactivation of a ~3542...ß-MHC GATA Site Can...DiscussionReferences |
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Northern blot analysis of 10 µg total RNA was performed as previously described.21 An isoform specific antisense deoxyoligonucleotide complementary to nucleotides 5846 to 5869 of the rat 3' untranslated region33 was used to detect ß-MHC mRNA as described previously.21 To detect ANF mRNA, a cDNA probe consisting of the PstI fragment of pANF-1,34 which encompasses rat ANF coding region (nucleotides 1 to 580), was radiolabelled by random priming and hybridized in 5x SSC (20x SSC=3 M NaCl, 0.3 M sodium citrate), 1x Denhardt's solution (Ficoll 200 µg/ml, polyvinylpyrrolidone 200 µg/ml, and bovine serum albumin 200 µg/ml), 50 mmol/L sodium phosphate (pH 7.2), 0.2% SDS, and 200 µg/ml denatured salmon sperm DNA at 65°C overnight and then washed in 2x SSC, 0.2% SDS at 65°C for 15 minutes. To normalize for loading and transfer, blots were hybridized with a 5'end-labeled deoxyoligonucleotide probe specific for 28S rRNA as described.35 Abundance of mRNAs were quantified by Phosphorimager analysis (Molecular Dynamics, Sunnyvale, CA). Values of ß-MHC and ANF mRNA were normalized to 28S rRNA.
Electrophoretic Mobility Shift Assays
Double-stranded oligonucleotides were designed
that contained GATA motifs from the ß-MHC or B-type
natriuretic peptide (BNP) promoters. The sequences of the
sense strand of these oligonucleotides were as follows:
ß-MHC GATA:
5'-AATGTAAGGGATATTTTTGCTTCACTTTGAG-3';36 ß-MHC
GATAmut: 5'-AATGTAAGGtcaATTTTTGCTTCACTTTGAG-3' (mutations in
small letters); BNP GATA:
5'-TGTGTCTGATAAATCAGAGATAACCCA-3';37 nonspecific:
AGAGCATTTTTGTTGGAGT-3'. Oligonucleotides were
synthesized by Life Technology Inc. Nuclear Extracts were prepared from
adult hearts of sham or aortic constricted rats and gel shift assays
were performed as described earlier.38 Probes
were used at 40 fmoles/reaction. In vitro translated GATA-4 was
prepared as previously described.38 In
competition experiments, all competitors were added at an 800-fold
molar excess. Anti-GATA-4 antiserum was kindly provided by Dr. David B.
Wilson (Washington University, St. Louis).39
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Statistical Analyses |
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TopAbstractIntroductionMaterials and MethodsAnimal Surgery and Tissue...RNA AnalysisStatistical AnalysesResultsTransactivation of a ~3542...ß-MHC GATA Site Can...DiscussionReferences |
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All data are expressed as means±SEM. The significance of differences between mean values was evaluated by the two tailed Student's t-test and differences considered significant at the P<.05 level.
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Results |
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TopAbstractIntroductionMaterials and MethodsAnimal Surgery and Tissue...RNA AnalysisStatistical AnalysesResultsTransactivation of a ~3542...ß-MHC GATA Site Can...DiscussionReferences |
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Transactivation of a ~3542 bp ß-MHC Promoter by Aortic Constriction |
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TopAbstractIntroductionMaterials and MethodsAnimal Surgery and Tissue...RNA AnalysisStatistical AnalysesResultsTransactivation of a ~3542...ß-MHC GATA Site Can...DiscussionReferences |
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To determine whether hemodynamic overload stimulates the transcriptional activities of the upstream regulatory sequences of ß-MHC and ANF genes, we evaluated the expression of luciferase reporter genes driven by 3542 bp of rat ß-MHC or 3412 bp of rat ANF 5' flanking sequences in control and hemodynamically overloaded ventricles. Gene transfer was accomplished by directly injecting plasmids p-3542ß-MHCluc or p-3412ANFluc into the left ventricular myocardium of the intact rats. In each case, a small quantity of a second plasmid, pRSVCAT was co-injected as an internal control for transfection efficiency and generalized changes in gene expression between the basal and induced states. On the day following gene transfer, animals were subjected to sham operation or to aortic constriction. Twelve days later, luciferase and CAT activities and steady state levels of the endogenous ß-MHC and ANF mRNAs were assessed in left ventricular homogenates.
As shown in Fig 1A
, the mean ratio of
left ventricular weight to body weight (LV/BW) was 71%
higher in aortic constricted as compared with sham operated animals
(P<.001). Among 14 aortic constricted animals, only one was
excluded from analysis due to insufficient left
ventricular hypertrophy, defined arbitrarily as
an increase in LV/BW < 30%. The mean levels of
endogenous ß-MHC and ANF mRNAs (normalized to those of
28S rRNA) were 3.2-fold (P<.001) and 10-fold
(P<.001) higher respectively in the left ventricles of
aortic constricted as compared with sham operated animals (Fig 1B).
Thus, this model of aortic constriction resulted in marked left
ventricular hypertrophy and induction in the
expression of ß-MHC and ANF genes.
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In animals injected with p-3542ß-MHCluc, the mean ratio of luciferase
activity to CAT activity (luc/CAT) was 3.0-fold higher in the left
ventricles of animals subjected to aortic constriction as compared with
sham operation (P<.005; Fig 1C
). This effect cannot be
attributed to differential effects of hemodynamic
overload on the posttranscriptional handling of luc or CAT mRNA or
protein because luc/CAT was similar in the left ventricles of aortic
constricted and sham operated animals co-injected with pRSVluc and
pRSVCAT (Fig 1C). Therefore, increases in the expression of
p-3542ß-MHCluc resulting from aortic constriction are due to
augmentation of the transcriptional activity of these ß-MHC promoter
sequences. This effect is sequence-specific as illustrated by the
absence of hemodynamic overload-stimulated increases in
transcription directed by the herpes simplex virus thymidine kinase
promoter (Fig 3), and a ß-MHC promoter deletion mutant
(p-203ß-MHCluc, Fig 2B). Thus, 3542 bp
of rat ß-MHC upstream sequence contain sufficient information to
increase transcription in response to aortic constriction to a
magnitude similar to that exhibited by steady state levels of
endogenous ß-MHC mRNA.
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We also evaluated the hemodynamic
overload-responsiveness of a promoter consisting of 3412 bp of rat ANF
5' flanking region. Although hemodynamic overload
results in marked ventricular expression of the
endogenous ANF gene, experiments in transgenic mice
harboring reporter constructs driven by various rat ANF sequences have
demonstrated the absence of inducible reporter expression in left
ventricles following aortic constriction.40
Injection of p-3412ANFluc resulted in similar luc/CAT in the left
ventricles of sham operated and aortic constricted rats (Fig 1C)
despite the 10-fold increase in steady state levels of
endogenous ANF mRNA in these ventricles (Fig 1B). The
fidelity of the construct itself was confirmed by demonstrating a
5.2±0.3-fold induction in reporter expression in response to 100 µM
of phenylephrine following transfection into cultured
neonatal rat cardiac myocytes (data not shown). Therefore, the results
of the gene injection experiments are in agreement with those obtained
in transgenic mice and demonstrate the inability of these
3412bp of rat ANF promoter to mediate hemodynamic
overload responsive expression of ANF gene in the rat
ventricular myocardium.
Analysis of the ß-MHC Promoter
To identify which DNA sequences mediate
hemodynamic overload stimulated increases in ß-MHC
transcription, nested 5' deletions were created in the rat 3542 bp
ß-MHC promoter. Promoters with 5' termini ranging from -3542 to -203
upstream of the ß-MHC cap site resulted in easily detectable amounts
of luciferase in adult cardiac myocytes in vivo (Fig 2A). The ability
of each construct to respond to hemodynamic overload
was evaluated in a series of experiments in which its transcriptional
activities in sham operated and aortic constricted hearts were compared
side by side according to the protocol described above (Fig 2B). For
each construct tested, the mean LV/BW was 60% higher in aortic
constricted as compared with sham operated animals indicating the
adequacy of the hemodynamic overload stimulus.
Deletions to -303 resulted in no significant decrease in inducibility,
demonstrating that sequences within the p-303ß-MHCluc construct are
sufficient to drive inducible expression. In addition, p-303ß-MHCluc
showed the highest level of inducibility among the five constructs
tested, suggesting that important elements which mediate inducible
expression lie 3' to -303. In contrast, deletion from -303 to -203
resulted in complete loss of inducible expression. This result cannot
be attributed to a loss of basal transcriptional activity as
p-203ß-MHCluc expressed levels of luciferase in the basal state which
exceeded those of another ß-MHC construct, p-1145ß-MHCluc, which
remained inducible. Thus, sequences -303/-203 are necessary for
hemodynamic overload-stimulated increases in the
transcription of p-303ß-MHCluc.
To determine the ability of sequences -303/-197 to confer
aortic constriction-inducible expression onto a heterologous neutral
promoter in the absence of other ß-MHC sequences, a construct
containing a single copy of these sequences fused in the forward
orientation to sequences -109 to +56 from the herpes simplex virus
thymidine kinase gene (p-303/-197tkCAT) was assessed (Fig 3). While the transcriptional activity of
the parental plasmid p-tkCAT was similar in control and
hemodynamically overloaded left ventricles, that of
p-303/-197tkCAT was 70% higher in the left ventricles of aortic
constricted as compared with sham operated animals
(P<.005). Thus, rat ß-MHC sequences -303/-197 are
sufficient to convert a neutral promoter to one which is
hemodynamic overload-responsive.
Role of M-CAT and GATA Elements
Sequences -303/-197 of the rat ß-MHC promoter contain M-CAT and
GATA motifs (Fig 4A). M-CAT elements play
a role in cardiac and skeletal muscle-specific expression in many
striated muscle genes including cardiac troponin
T,41 skeletal
-actin,12,13 -MHC,42
as well as ß-MHC11,43–45 (reviewed in ref.
46). Moreover, the proximal M-CAT element in the rat ß-MHC gene has
been shown to be both necessary and sufficient for transactivation of
this gene during hypertrophy induced by
1-adrenergic agonists in cultured neonatal
cardiac myocytes.11 To test the role of M-CAT
elements in hemodynamic overload-induced activation of
the ß-MHC promoter in the setting of adult cardiac myocytes in vivo,
mutations previously demonstrated to abolish the binding of nuclear
proteins11,47 were introduced in the context of
rat ß-MHC sequences -333 to +34 (p-333mutA+Bß-MHCluc). Both M-CAT
elements were mutated simultaneously to eliminate the
possibility of redundancy. The expression of this construct was
compared with that of the corresponding wild type construct
(p-333wtß-MHCluc) in the left ventricles of sham operated and aortic
constricted rats. As shown in Fig 4B, mutation of M-CAT elements
decreased transcriptional activity in sham operated ventricles by 82%
(P<.005). In contrast, expression of both p-333wtß-MHCluc
and p-333mutA+Bß-MHCluc were induced 3.5-fold by aortic constriction
(P<.005 for both; Fig 4C). Thus, in the context of the 333
bp ß-MHC promoter, M-CAT elements are required for full levels of
transcription in the basal state but not for transcription induced by
hemodynamic overload.
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We next examined the role of the GATA motif. GATA elements have been
shown to be important for cardiac-specific transcription in the context
of many other cardiac genes including -MHC,38
cardiac troponin T,39 myosin light chain
1/348 and B-type natriuretic peptide
(BNP).37 A mutation in the core GATA sequence
that was previously demonstrated to abolish binding of nuclear proteins
in other genes was studied in the context of -333 bp ß-MHC promoter
(p-333mutGATAß-MHCluc). While this mutation did not affect basal
transcription (Fig 4B), it markedly reduced hemodynamic
overload-inducible transcription (1.6-fold, NS for
p-333mutGATAß-MHCluc as compared with 3.5-fold,
P<.005 for p-333wtß-MHCluc; Fig 4C). These findings
indicate that the GATA sequences play a role in mediating the induction
of ß-MHC expression during hemodynamic overload
hypertrophy in vivo.
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ß-MHC GATA Site Can Interact with GATA-4 in Aortic Constricted Hearts |
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TopAbstractIntroductionMaterials and MethodsAnimal Surgery and Tissue...RNA AnalysisStatistical AnalysesResultsTransactivation of a ~3542...ß-MHC GATA Site Can...DiscussionReferences |
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To determine if the GATA motif in the ß-MHC promoter can interact specifically with GATA-4, electrophoretic mobility shift assays (EMSA) were performed. In vitro translated GATA-4 was probed with a radiolabeled oligonucleotide containing the ß-MHC GATA site in the presence or absence of competitor DNAs (Fig 5A
, lanes 1 to 4). Among three retarded
bands, competition EMSAs revealed that only the slowest migrating band
represents GATA sequence-specific binding as evidenced by the
fact that it was competed by an excess of unlabeled ß-MHC GATA
oligonucleotide (lane 2) or by an
oligonucleotide containing a previously demonstrated
GATA site in the BNP promoter (lane 3), but not by the same amount of a
nonspecific oligonucleotide (lane 4) or by an
oligonucleotide containing the ß-MHC GATA site into
which point mutations (Fig 4A) that ablate hemodynamic
overload responsiveness had been introduced (data not shown). In
addition, the retarded band represents an interaction of the
probe with GATA-4 because it was absent in unprogrammed lysate (data
not shown). To compare the mobility of this band with complexes formed
on an established GATA site, in vitro translated GATA-4 was probed with
an oligonucleotide encompassing the BNP GATA motif (Fig 5A, lanes 5 to 8). Two GATA sequence specific interactions were
observed with the slower one similar in mobility to that formed on the
ß-MHC GATA site (compare lanes 1 and 5). Competitions with an excess
of unlabeled BNP GATA competed both this slower band as well as a
faster migrating complex (lane 6). In contrast, the same amount of
ß-MHC GATA oligonucleotide competed only the slower
complex (lane 7), while nonspecific oligonucleotide
competed neither band (lane 8). Of note, the intensity of the slow
mobility complex increased when in vitro translated GATA-4 was
incubated for 72 hours at 4°C prior to use compared
with freshly prepared protein (data not shown). Thus, these data
demonstrate that the ß-MHC GATA site can interact specifically with
GATA-4 in vitro and the mobility of the resulting band is similar to
that of the slower of the two bands resulting from the specific
interaction of GATA-4 with the BNP GATA site. These data also suggest
that GATA-4 may bind the ß-MHC GATA site as a multimer.
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To determine if the ß-MHC GATA site binds GATA proteins in the
context of the normal and hemodynamically overloaded
adult myocardium, EMSAs were performed with nuclear
extracts from sham operated and aortic constricted hearts. Multiple
bands were observed following the incubation of sham operated nuclear
extract with ß-MHC GATA-4. Competition studies revealed that all of
these protein-DNA interactions were nonspecific (data not shown). With
aortic constricted extract, multiple nonspecific bands were observed as
well as one band representing GATA-specific binding (data
not shown). The intensity of the latter was quite faint, however,
precluding supershift experiments to identify the component protein(s).
This is not surprising given the the results of the studies using in
vitro translated GATA-4 (Fig 5A) suggesting that the ß-MHC GATA site
has a relatively low affinity for GATA-4. Therefore, to circumvent this
problem and to test whether the ß-MHC GATA site could interact with
GATA-4 in the context of an aortic constricted nuclear extract, we used
the BNP oligonucleotide as a probe (Fig 5B). Several
bands were observed when aortic constricted heart extract was probed
with the BNP GATA. Addition of preimmune serum to the reaction did not
significantly affect the interactions (lane 1). In contrast, the
addition of GATA-4 antiserum resulted in a supershift, establishing
that GATA-4 is one of the components (lane 2). This supershift was
competed by unlabeled ß-MHC GATA oligonucleotide
(lane 3) but not by an oligonucleotide identical except
for point mutations in the ß-MHC GATA site (Fig 4A) that ablate
hemodynamic overload responsiveness (lane 4). These
data indicate that the ß-MHC GATA site can bind in a sequence
specific manner to GATA-4 in nuclear extract from aortic constricted
hearts.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsAnimal Surgery and Tissue...RNA AnalysisStatistical AnalysesResultsTransactivation of a ~3542...ß-MHC GATA Site Can...DiscussionReferences |
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A combination of humoral, mechanical, and neural factors, referred to collectively as hemodynamic overload, plays a key role in the pathogenesis of virtually all disorders of heart muscle. This stimulus elicits hypertrophy of cardiac myocytes, characterized by increases in cell volume and the synthesis of additional sarcomeres. After a highly variable time interval, these events are often followed by the deterioration of contractile function. A long sought after goal has been to understand the molecular basis by which hemodynamic overload elicits these pathological effects. Since the expression patterns of specific cardiac genes change in temporal association with these events,1–8 it has been postulated that the signaling pathways that control the expression of these genes overlap significantly with those that mediate these structural and functional changes. For this reason, significant efforts have been directed toward understanding the regulation of these genes both in the basal state and in response to various perturbations.9–16 These studies, however, have been limited largely to cultured cardiac myocytes from neonatal rats and to purely humoral or relatively simple mechanical stimuli that can be replicated in a culture dish. We have established a system that permits one to delineate the mechanisms by which hemodynamic overload, in all its complexity, modulates gene expression during hypertrophy of adult cardiac myocytes in vivo. This system has been employed to study the regulation of the ß-MHC gene, whose accumulation in myocytes of adult rodent ventricles closely parallels the development of cardiac hypertrophy following aortic constriction.4,8 These experiments demonstrate that induction of ß-MHC expression during hemodynamic overload hypertrophy is regulated at the level of transcription and that a GATA element within ß-MHC sequences -303/-197 plays a role in mediating this response.
Direct evidence for the transcriptional regulation of cardiac genes
during hypertrophy has been provided by nuclear run-on
assays evaluating the response of the -skeletal actin gene to
1-adrenergic agonists in cultured neonatal
cardiac myocytes.49 In addition, transient
transfections of reporter constructs into these cells have provided
indirect evidence that transcription plays a role in the increases in
ß-MHC,11 ventricular myosin light
chain 2,9,14 and ANF9,10
expression in response to 1-adrenergic
agonists9,11,14 and
endothelin-1.10 Although nuclear run-on assays
have been successfully performed using neonatal cardiac
tissue,50 the difficulty in isolating
transcriptionally active nuclei from adult cardiac tissue has impeded
direct evaluation of the effect of hemodynamic overload
on the transcription of specific genes in the context of the intact
adult heart. The reporter assays in this study provide the first
evidence, albeit indirect, that hemodynamic overload
increases ß-MHC transcription. Moreover, the quantitatively similar
increments in the transcriptional activities of ß-MHC regulatory
sequences and in the steady state levels of endogenous
ß-MHC mRNA suggest that transcriptional regulation plays the
predominant role in the stimulation of ß-MHC expression by
hemodynamic overload. Interestingly, the expression of
a reporter gene driven by 3412 bp of ANF upstream sequence was not
increased by hemodynamic overload despite marked
induction in the expression of the endogenous ANF gene.
This result, which is consistent with the findings of
transgenic experiments,40 suggests that
additional cis-acting sequences are required for
hemodynamic overload-stimulated ANF transcription
and/or increases in endogenous ANF mRNA levels occur
through posttranscriptional mechanisms.
The transcriptional activities of nested 5' ß-MHC promoter deletions to -303 retained full hemodynamic overload-respnsiveness. In contrast, hemodynamic overload did not induce the expression of a transcriptionally active mutant with 5' terminus at -203. Although these findings by no means exclude the existence of hemodynamic overload-responsive sequences elsewhere in the rat ß-MHC gene, they demonstrate clearly that sequences -303/-203 are necessary for hemodynamic overload-inducibility in the context of the -303 mutant. In addition, although not as efficient as in its normal context, a single copy of the 107-bp fragment, -303/-197, was able to confer hemodynamic overload-responsiveness onto a heterologous neutral promoter. Thus, these sequences are sufficient to transduce signals generated by hemodynamic overload in adult cardiac myocytes. Taken together, these findings suggest that sequences -303/-197 are involved in mediating hemodynamic overload-stimulated transcription of the ß-MHC gene.
The M-CAT motif is present in the promoters of several striated muscle-specific genes where it functions as a positive regulatory element.41–46 Rat ß-MHC sequences -303/-197 contain two such M-CAT elements: distal (-274 to -280) and proximal (-204 to -210). The distal M-CAT element in the ß-MHC promoter is perfectly conserved among human, rabbit, rat, and mouse and the proximal one, while not conserved, matches the M-CAT consensus sequence exactly.11,42–45,51 Both elements have been shown to be binding sites for the transcription factor TEF-1.42,47,52 Mutation of either element in the context of a truncated ß-MHC promoter diminishes transcription both in cultured cardiac myocytes11,42–44 and in the hearts of adult transgenic mice.45 Our observation that simultaneous disruption of both M-CAT elements in the setting of a 333 bp ß-MHC promoter markedly decreases transcriptional activity is consistent with this data.41–46 These mutations have no effect on basal transcriptional activity of a ~5000 bp mouse ß-MHC promoter in transgenic mice, however, suggesting that loss of both M-CAT elements can be compensated for by upstream sequences.45 Thus, while not indispensable, M-CAT elements appear to play a role in basal transcription of the ß-MHC gene.
Previous work in cell culture models of neonatal cardiac myocyte
hypertrophy has delineated potential elements which mediate
1-adrenergic-, endothelin-1-, or transforming
growth factor-ß-inducible expression during
hypertrophy.11–14,16 These include
HF-1 in the ventricular myosin light chain
promoter,14 GAG motif in the ANF
promoter,16 and M-CAT elements in the
-skeletal actin and ß-MHC promoters.11–13
The M-CAT element has also been shown to be necessary and sufficient
for 1-adrenergic-stimulated transcription of
the ß-MHC gene and necessary for
1-adrenergic and transforming growth
factor-ß-stimulated activation of the -skeletal actin promoter
during hypertrophy in cultured neonatal cardiac
myocytes.11–13 In contrast with these in vitro
studies, simultaneous mutations in both M-CAT elements,
adequate to abrogate binding of nuclear proteins and destroy enhancer
function, had no effect on the hemodynamic
overload-responsiveness of a 333 bp rat ß-MHC promoter in adult
cardiac myocytes in vivo. Thus, even in the setting of a truncated
promoter, M-CAT elements appear dispensable for
hemodynamic overload-induced transcription. Our data do
not rule out the possibility that M-CAT elements still contribute to
hemodynamic overload responsiveness, however. In
addition, our experiments were limited to a single time point
relatively late after the onset of hemodynamic
overload; therefore, it remains possible that M-CAT elements contribute
to inducible expression at earlier points in time.
Rat ß-MHC sequences -303/-197 also contain a putative GATA element.
GATA factors 4, 5, and 6, which are present in cardiac myocytes
and/or their progenitors, play important roles in the transcriptional
regulation of cardiac genes (reviewed in ref. 53) and heart
morphogenesis.54,55 Although mutation of GATA
elements in -MHC,38 cardiac troponin
T,39 myosin light chain
1/348 and BNP37 diminishes
their transcriptional activities in cardiac myocytes in
culture37,39 and in
vivo,38,48 it had no effect on that of the 333 bp
ß-MHC promoter. In contrast, this same GATA mutation markedly
attenuated the ability of the ß-MHC promoter to respond to
hemodynamic overload. We hasten to point out, however,
that although this element clearly mediates the transcriptional
response of ß-MHC to hemodynamic overload, it may not
be the only element in the -303/-203 ß-MHC fragment that regulates
this response. Indeed, given the potential complexities of the
hemodynamic overload stimulus and the multitude of
pathways that mediate it, it would not be surprising if combinatorial
interactions among several elements were required to precisely
reconstitute inducible expression during hypertrophy in
vivo. In any event, the differential effects of M-CAT or GATA mutations
on basal and hemodynamic overload-induced transcription
suggest that these processes are mediated, at least in part, through
different mechanisms.
The GATA element has been shown to be a binding site of GATA transcription factors in the context of other cardiac genes37–39,48 and the EMSA studies herein demonstrate that ß-MHC GATA site can also bind to GATA-4 both in vitro and in vivo. Interestingly, the mobility of the complex resulting from the interaction between ß-MHC GATA site and in vitro translated GATA-4 was similar to that of one of the complexes produced by the binding of the BNP GATA site to GATA-4. This complex was less abundant and of slower mobility than the major complex formed between the BNP GATA site and GATA-4. This result suggests that GATA-4 may be binding the ß-MHC GATA site as a higher ordered complex (possibly a dimer) as compared with the majority of its binding to the BNP GATA site (possibly a monomer). Although this interpretation is provisional and needs to be tested directly, it suggests one model by which GATA transcription factors could modulate the basal transcription of certain cardiac genes and the inducible expression of others. In this model, the promoters dependent on GATA for basal transcriptional activity would be activated by the binding of GATA monomers while the higher concentration of GATA factors in the hemodynamically overloaded state38 would drive the formation of multimeric complexes on GATA sites that mediate hemodynamic overload inducible transcription. To understand the precise mechanism by which GATA sequences mediate hemodynamic overload responsive transcription, however, it will be necessary to define exactly which GATA, as well as possibly nonGATA, proteins bind these sequences and the stoichiometry of these interactions in control and hemodynamically overloaded hearts.
The results of our experiments differ in important respects from those
obtained in various cell culture models of cardiac myocyte
hypertrophy. For example, although a ~3000 bp rat ANF
promoter is activated by 1-adrenergic
agonists and endothelin-1 in cultured rat neonatal cardiac myocytes
(9,10; and our data not shown), its transcriptional activity is not
increased by hemodynamic overload following injection
into adult rat hearts (Fig 1C) or in transgenic
mice.40 Conversely, although rat ß-MHC
promoters longer than 303 bp do respond to hemodynamic
overload in adult hearts in vivo, even a 673 bp promoter did not
respond to stretch-induced hypertrophy in cultured neonatal
cardiac myocytes.15 These discrepancies support
the notion that differences exist between the pathways that mediate
induction of ß-MHC and ANF expression during hypertrophy
due to chronic hemodynamic overload in intact adult
animals and short-term stimuli in cultured neonatal cardiac myocytes.
These discrepancies may be attributable to differences in the
developmental stage of cells (adult versus neonatal), the extracellular
environment (in vivo vs. in vitro), or the nature of the hypertrophic
stimulus itself (hemodynamic overload versus a single
humoral stimulus or stretch). Because cultured neonatal cardiac
myocytes provide a relatively simple means of testing a single well
defined stimulus in the absence of other confounding factors, they have
provided and will undoubtedly continue to provide an important
experimental approach for understanding cardiac
hypertrophy. Nevertheless, the differences noted above
underscore the importance of eventually testing all conclusions in
vivo.
Note Added in Proof
While this manuscript was under review, Herzig et
al.56 reported that GATA-4 is involved in induction of the
expression of the angiotensin II type 1a receptor gene in response to
hemodynamic overload.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by grants from the NIH (HL-02699 to Dr Kitsis; HL-43662 to Dr Markham), and from the American Heart Association, New York City Affiliate (Dr Kitsis). Dr Kitsis is the Charles and Tamara Krasne Faculty Scholar in Cardiovascular Research of the Albert Einstein College of Medicine. We thank Dr Francis Siri for advice regarding the aortic constriction model.
Received July 24, 1997; revision received September 5, 1997; accepted September 25, 1997.
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References |
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 S. Miyamoto, T. Kawamura, T. Morimoto, K. Ono, H. Wada, Y. Kawase, A. Matsumori, R. Nishio, T. Kita, and K. Hasegawa Histone Acetyltransferase Activity of p300 Is Required for the Promotion of Left Ventricular Remodeling After Myocardial Infarction in Adult Mice In Vivo Circulation, February 7, 2006; 113(5): 679 - 690. [Abstract] [Full Text] [PDF]
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 T. Oka, Y.-S. Dai, and J. D. Molkentin Regulation of Calcineurin through Transcriptional Induction of the calcineurin A{beta} Promoter In Vitro and In Vivo Mol. Cell. Biol., August 1, 2005; 25(15): 6649 - 6659. [Abstract] [Full Text] [PDF]
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I. S. Kathiriya, I. N. King, M. Murakami, M. Nakagawa, J. M. Astle, K. A. Gardner, R. D. Gerard, E. N. Olson, D. Srivastava, and O. Nakagawa Hairy-related Transcription Factors Inhibit GATA-dependent Cardiac Gene Expression through a Signal-responsive Mechanism J. Biol. Chem., December 24, 2004; 279(52): 54937 - 54943. [Abstract] [Full Text] [PDF]
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S. Mathew, E. Mascareno, and M. A. Q. Siddiqui A Ternary Complex of Transcription Factors, Nished and NFATc4, and Co-activator p300 Bound to an Intronic Sequence, Intronic Regulatory Element, Is Pivotal for the Up-regulation of Myosin Light Chain-2v Gene in Cardiac Hypertrophy J. Biol. Chem., September 24, 2004; 279(39): 41018 - 41027. [Abstract] [Full Text] [PDF]
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M. Hirai, K. Ono, T. Morimoto, T. Kawamura, H. Wada, T. Kita, and K. Hasegawa FOG-2 Competes with GATA-4 for Transcriptional Coactivator p300 and Represses Hypertrophic Responses in Cardiac Myocytes J. Biol. Chem., September 3, 2004; 279(36): 37640 - 37650. [Abstract] [Full Text] [PDF]
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K. J. A. McCullagh, E. Calabria, G. Pallafacchina, S. Ciciliot, A. L. Serrano, C. Argentini, J. M. Kalhovde, T. Lomo, and S. Schiaffino NFAT is a nerve activity sensor in skeletal muscle and controls activity-dependent myosin switching PNAS, July 20, 2004; 101(29): 10590 - 10595. [Abstract] [Full Text] [PDF]
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O. Tenhunen, B. Sarman, R. Kerkela, I. Szokodi, L. Papp, M. Toth, and H. Ruskoaho Mitogen-activated Protein Kinases p38 and ERK 1/2 Mediate the Wall Stress-induced Activation of GATA-4 Binding in Adult Heart J. Biol. Chem., June 4, 2004; 279(23): 24852 - 24860. [Abstract] [Full Text] [PDF]
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 J. L. Hall, S. Grindle, X. Han, D. Fermin, S. Park, Y. Chen, R. J. Bache, A. Mariash, Z. Guan, S. Ormaza, et al. Genomic profiling of the human heart before and after mechanical support with a ventricular assist device reveals alterations in vascular signaling networks Physiol Genomics, May 19, 2004; 17(3): 283 - 291. [Abstract] [Full Text] [PDF]
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A. Aries, P. Paradis, C. Lefebvre, R. J. Schwartz, and M. Nemer Essential role of GATA-4 in cell survival and drug-induced cardiotoxicity PNAS, May 4, 2004; 101(18): 6975 - 6980. [Abstract] [Full Text] [PDF]
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R. B. Vega, R. Bassel-Duby, and E. N. Olson Control of Cardiac Growth and Function by Calcineurin Signaling J. Biol. Chem., September 26, 2003; 278(39): 36981 - 36984. [Full Text] [PDF]
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S. Pikkarainen, H. Tokola, T. Majalahti-Palviainen, R. Kerkela, N. Hautala, S. S. Bhalla, F. Charron, M. Nemer, O. Vuolteenaho, and H. Ruskoaho GATA-4 Is a Nuclear Mediator of Mechanical Stretch-activated Hypertrophic Program J. Biol. Chem., June 20, 2003; 278(26): 23807 - 23816. [Abstract] [Full Text] [PDF]
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H. Akazawa and I. Komuro Roles of Cardiac Transcription Factors in Cardiac Hypertrophy Circ. Res., May 30, 2003; 92(10): 1079 - 1088. [Abstract] [Full Text] [PDF]
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T. Yanazume, K. Hasegawa, T. Morimoto, T. Kawamura, H. Wada, A. Matsumori, Y. Kawase, M. Hirai, and T. Kita Cardiac p300 Is Involved in Myocyte Growth with Decompensated Heart Failure Mol. Cell. Biol., May 15, 2003; 23(10): 3593 - 3606. [Abstract] [Full Text] [PDF]
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 Q. He, M. Mendez, and M. C. LaPointe Regulation of the human brain natriuretic peptide gene by GATA-4 Am J Physiol Endocrinol Metab, July 1, 2002; 283(1): E50 - E57. [Abstract] [Full Text] [PDF]
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S. E. Hardt and J. Sadoshima Glycogen Synthase Kinase-3{beta}: A Novel Regulator of Cardiac Hypertrophy and Development Circ. Res., May 31, 2002; 90(10): 1055 - 1063. [Abstract] [Full Text] [PDF]
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R. Kerkela, S. Pikkarainen, T. Majalahti-Palviainen, H. Tokola, and H. Ruskoaho Distinct Roles of Mitogen-activated Protein Kinase Pathways in GATA-4 Transcription Factor-mediated Regulation of B-type Natriuretic Peptide Gene J. Biol. Chem., April 12, 2002; 277(16): 13752 - 13760. [Abstract] [Full Text] [PDF]
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T. Yanazume, K. Hasegawa, H. Wada, T. Morimoto, M. Abe, T. Kawamura, and S. Sasayama Rho/ROCK Pathway Contributes to the Activation of Extracellular Signal-regulated Kinase/GATA-4 during Myocardial Cell Hypertrophy J. Biol. Chem., March 1, 2002; 277(10): 8618 - 8625. [Abstract] [Full Text] [PDF]
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W. Zhang Old and new tools to dissect calcineurin's role in pressure-overload cardiac hypertrophy Cardiovasc Res, February 1, 2002; 53(2): 294 - 303. [Abstract] [Full Text] [PDF]
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Q. Liang, R. J. Wiese, O. F. Bueno, Y.-S. Dai, B. E. Markham, and J. D. Molkentin The Transcription Factor GATA4 Is Activated by Extracellular Signal-Regulated Kinase 1- and 2-Mediated Phosphorylation of Serine 105 in Cardiomyocytes Mol. Cell. Biol., November 1, 2001; 21(21): 7460 - 7469. [Abstract] [Full Text] [PDF]
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 M. Marttila, N. Hautala, P. Paradis, M. Toth, O. Vuolteenaho, M. Nemer, and H. Ruskoaho GATA4 Mediates Activation of the B-Type Natriuretic Peptide Gene Expression in Response to Hemodynamic Stress Endocrinology, November 1, 2001; 142(11): 4693 - 4700. [Abstract] [Full Text] [PDF]
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 F. Charron, G. Tsimiklis, M. Arcand, L. Robitaille, Q. Liang, J. D. Molkentin, S. Meloche, and M. Nemer Tissue-specific GATA factors are transcriptional effectors of the small GTPase RhoA Genes & Dev., October 15, 2001; 15(20): 2702 - 2719. [Abstract] [Full Text] [PDF]
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C. E. Wright, P. W. Bodell, F. Haddad, A. X. Qin, and K. M. Baldwin In vivo regulation of the {beta}-myosin heavy chain gene in hypertensive rodent heart Am J Physiol Cell Physiol, May 1, 2001; 280(5): C1262 - C1276. [Abstract] [Full Text] [PDF]
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M. Flesch On the trail of cardiac specific transcription factors Cardiovasc Res, April 1, 2001; 50(1): 3 - 6. [Full Text] [PDF]
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N. Hautala, H. Tokola, M. Luodonpaa, J. Puhakka, H. Romppanen, O. Vuolteenaho, and H. Ruskoaho Pressure Overload Increases GATA4 Binding Activity via Endothelin-1 Circulation, February 6, 2001; 103(5): 730 - 735. [Abstract] [Full Text] [PDF]
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Q. He, D. Wang, X.-P. Yang, O. A. Carretero, and M. C. LaPointe Inducible regulation of human brain natriuretic peptide promoter in transgenic mice Am J Physiol Heart Circ Physiol, January 1, 2001; 280(1): H368 - H376. [Abstract] [Full Text] [PDF]
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E. Oie, R. Bjornerheim, O. P. F. Clausen, and H. Attramadal Cyclosporin A inhibits cardiac hypertrophy and enhances cardiac dysfunction during postinfarction failure in rats Am J Physiol Heart Circ Physiol, June 1, 2000; 278(6): H2115 - H2123. [Abstract] [Full Text] [PDF]
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W. Zhu, Y. Zou, I. Shiojima, S. Kudoh, R. Aikawa, D. Hayashi, M. Mizukami, H. Toko, F. Shibasaki, Y. Yazaki, et al. Ca2+/Calmodulin-dependent Kinase II and Calcineurin Play Critical Roles in Endothelin-1-induced Cardiomyocyte Hypertrophy J. Biol. Chem., May 12, 2000; 275(20): 15239 - 15245. [Abstract] [Full Text] [PDF]
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T. Morimoto, K. Hasegawa, S. Kaburagi, T. Kakita, H. Wada, T. Yanazume, and S. Sasayama Phosphorylation of GATA-4 Is Involved in alpha 1-Adrenergic Agonist-responsive Transcription of the Endothelin-1 Gene in Cardiac Myocytes J. Biol. Chem., April 28, 2000; 275(18): 13721 - 13726. [Abstract] [Full Text] [PDF]
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Y. Nagatomo, B. A. Carabello, M. Hamawaki, S. Nemoto, T. Matsuo, and P. J. McDermott Translational mechanisms accelerate the rate of protein synthesis during canine pressure-overload hypertrophy Am J Physiol Heart Circ Physiol, December 1, 1999; 277(6): H2176 - H2184. [Abstract] [Full Text] [PDF]
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T. Kakita, K. Hasegawa, T. Morimoto, S. Kaburagi, H. Wada, and S. Sasayama p300 Protein as a Coactivator of GATA-5 in the Transcription of Cardiac-restricted Atrial Natriuretic Factor Gene J. Biol. Chem., November 26, 1999; 274(48): 34096 - 34102. [Abstract] [Full Text] [PDF]
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L. Kim, T. Lee, J. Fu, and M. E. Ritchie Characterization of MAP kinase and PKC isoform and effect of ACE inhibition in hypertrophy in vivo Am J Physiol Heart Circ Physiol, November 1, 1999; 277(5): H1808 - H1816. [Abstract] [Full Text] [PDF]
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D. R. Vyas, J. J. McCarthy, and R. W. Tsika Nuclear Protein Binding at the beta -Myosin Heavy Chain A/T-rich Element Is Enriched following Increased Skeletal Muscle Activity J. Biol. Chem., October 22, 1999; 274(43): 30832 - 30842. [Abstract] [Full Text] [PDF]
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F. Charron, P. Paradis, O. Bronchain, G. Nemer, and M. Nemer Cooperative Interaction between GATA-4 and GATA-6 Regulates Myocardial Gene Expression Mol. Cell. Biol., June 1, 1999; 19(6): 4355 - 4365. [Abstract] [Full Text] [PDF]
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J. J. McCarthy, D. R. Vyas, G. L. Tsika, and R. W. Tsika Segregated Regulatory Elements Direct beta -Myosin Heavy Chain Expression in Response to Altered Muscle Activity J. Biol. Chem., May 14, 1999; 274(20): 14270 - 14279. [Abstract] [Full Text] [PDF]
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T. Morimoto, K. Hasegawa, S. Kaburagi, T. Kakita, H. Masutani, R. N. Kitsis, A. Matsumori, and S. Sasayama GATA-5 Is Involved in Leukemia Inhibitory Factor-responsive Transcription of the beta -Myosin Heavy Chain Gene in Cardiac Myocytes J. Biol. Chem., April 30, 1999; 274(18): 12811 - 12818. [Abstract] [Full Text] [PDF]
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G. Cheng, T. P. Hagen, M. L. Dawson, K. V. Barnes, and D. R. Menick The Role of GATA, CArG, E-box, and a Novel Element in the Regulation of Cardiac Expression of the Na+-Ca2+ Exchanger Gene J. Biol. Chem., April 30, 1999; 274(18): 12819 - 12826. [Abstract] [Full Text] [PDF]
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E. N. Olson and J. D. Molkentin Prevention of Cardiac Hypertrophy by Calcineurin Inhibition : Hope or Hype? Circ. Res., April 2, 1999; 84(6): 623 - 632. [Full Text] [PDF]
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P. H. Sugden Signaling in Myocardial Hypertrophy : Life After Calcineurin? Circ. Res., April 2, 1999; 84(6): 633 - 646. [Full Text] [PDF]
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W. Zhang, R. C. Kowal, F. Rusnak, R. A. Sikkink, E. N. Olson, and R. G. Victor Failure of Calcineurin Inhibitors to Prevent Pressure-Overload Left Ventricular Hypertrophy in Rats Circ. Res., April 2, 1999; 84(6): 722 - 728. [Abstract] [Full Text] [PDF]
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B. Ding, R. L. Price, T. K. Borg, E. O. Weinberg, P. F. Halloran, and B. H. Lorell Pressure Overload Induces Severe Hypertrophy in Mice Treated With Cyclosporine, an Inhibitor of Calcineurin Circ. Res., April 2, 1999; 84(6): 729 - 734. [Abstract] [Full Text] [PDF]
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T. I. Slepak, K. A. Webster, J. Zang, H. Prentice, A. O'Dowd, M. N. Hicks, and N. H. Bishopric Control of Cardiac-specific Transcription by p300 through Myocyte Enhancer Factor-2D J. Biol. Chem., March 2, 2001; 276(10): 7575 - 7585. [Abstract] [Full Text] [PDF]
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T. Morimoto, K. Hasegawa, H. Wada, T. Kakita, S. Kaburagi, T. Yanazume, and S. Sasayama Calcineurin-GATA4 Pathway Is Involved in beta -Adrenergic Agonist-responsive Endothelin-1 Transcription in Cardiac Myocytes J. Biol. Chem., September 7, 2001; 276(37): 34983 - 34989. [Abstract] [Full Text] [PDF]
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Q. Liang, L. J. De Windt, S. A. Witt, T. R. Kimball, B. E. Markham, and J. D. Molkentin The Transcription Factors GATA4 and GATA6 Regulate Cardiomyocyte Hypertrophy in Vitro and in Vivo J. Biol. Chem., August 3, 2001; 276(32): 30245 - 30253. [Abstract] [Full Text] [PDF]
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C. Morisco, K. Seta, S. E. Hardt, Y. Lee, S. F. Vatner, and J. Sadoshima Glycogen Synthase Kinase 3beta Regulates GATA4 in Cardiac Myocytes J. Biol. Chem., July 20, 2001; 276(30): 28586 - 28597. [Abstract] [Full Text] [PDF]
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Y.-S. Dai and B. E. Markham p300 Functions as a Coactivator of Transcription Factor GATA-4 J. Biol. Chem., September 28, 2001; 276(40): 37178 - 37185. [Abstract] [Full Text] [PDF]
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J. D. Molkentin The Zinc Finger-containing Transcription Factors GATA-4, -5, and -6. UBIQUITOUSLY EXPRESSED REGULATORS OF TISSUE-SPECIFIC GENE EXPRESSION J. Biol. Chem., December 8, 2000; 275(50): 38949 - 38952. [Full Text] [PDF]
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A. L. Serrano, M. Murgia, G. Pallafacchina, E. Calabria, P. Coniglio, T. Lomo, and S. Schiaffino Calcineurin controls nerve activity-dependent specification of slow skeletal muscle fibers but not muscle growth PNAS, November 6, 2001; 98(23): 13108 - 13113. [Abstract] [Full Text] [PDF]
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M. Asakawa, H. Takano, T. Nagai, H. Uozumi, H. Hasegawa, N. Kubota, T. Saito, Y. Masuda, T. Kadowaki, and I. Komuro Peroxisome Proliferator-Activated Receptor {gamma} Plays a Critical Role in Inhibition of Cardiac Hypertrophy In Vitro and In Vivo Circulation, March 12, 2002; 105(10): 1240 - 1246. [Abstract] [Full Text] [PDF]
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