Department of Physiology and Biophysics, University of California, Irvine, California 92697
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ABSTRACT |
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The main goal of this study was to examine the
transcriptional activity of different-length -myosin heavy chain
(
-MHC) promoters in the hypertensive rodent heart using the direct
gene transfer approach. A hypertensive state was induced by abdominal
aortic constriction (AbCon) sufficient to elevate mean arterial
pressure by ~45% relative to control. Results show that
-MHC
promoter activity of all tested wild-type constructs, i.e.,
3500,
408,
299,
215,
171, and
71 bp, was significantly increased in
AbCon hearts. In the normal control hearts, expression of the
71-bp construct was comparable to that of the promoterless vector, but its
induction by AbCon was comparable to that of the other constructs. Additional results, based on mutation analysis and DNA gel mobility shift assays targeting
e1,
e2, GATA, and
e3 elements, show that these previously defined cis-elements in the proximal
promoter are indeed involved in maintaining basal promoter activity;
however, none of these elements, either individually or collectively,
appear to be major players in mediating the hypertension response of the
-MHC gene. Collectively, these results indicate that three separate regions on the
-MHC promoter are involved in the induction of the gene in response to hypertension: 1) a distal region
between
408 and
3500 bp, 2) a proximal region between
299 and
215 bp, and 3) a basal region within
71 bp of
the transcription start site. Future research needs to further
characterize these responsive regions to more fully delineate
-MHC
transcriptional regulation in response to pressure overload.
hypertension; transcription; dual luciferase; in vivo gene transfer
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INTRODUCTION |
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ADULT MAMMALIAN
CARDIAC MUSCLE expresses two genes encoding for myosin heavy
chains (MHCs), which have been designated - and
-MHC
(28). The
- and
-MHC genes are members of the MHC multigene family, in which each of the genes is expressed and highly
regulated in a muscle-type-specific fashion (28, 30). Although
-MHC is expressed only in the heart,
-MHC is expressed in the heart and is also the major myosin isoform expressed in slow-twitch skeletal muscle (30).
In the myocardium, the MHC isoform composition affects the
physiodynamics and energetics of the working heart, which are of great
physiological significance to cardiac performance. The -MHC isoform
is characterized by a higher ATPase activity and faster shortening
speed than the
-MHC isoform (2, 21). Thus hearts rich
in the
-isoform have a high intrinsic contractility, whereas those
rich in the
-isoform have a lower contractility but a higher economy
of tension development (2). In the adult rodent, the
-MHC is the predominant isoform expressed in the ventricles, accounting for ~85-90% of the total MHC protein pool, whereas the
-MHC accounts for the remaining 10-15% (18).
In cardiac cells of different mammalian species, the expression of the
two MHCs is developmentally regulated (28) and can be
altered by a variety of pathophysiological conditions, including
abnormal thyroid status (14, 24, 28, 38), diabetes
(5), and hemodynamic overload (22, 29).
Chronic hemodynamic overload, as occurs in hypertension, is a complex
physiological stimulus that triggers significant changes in myocardial
structure and function. The visible changes include cardiac
hypertrophy, which is expressed as an increased heart weight-to-body
weight ratio, while changes on the molecular level include altered
phenotype expression of specific cardiac genes that include several
contractile and regulatory proteins (4, 25, 31, 39). For
example, in the hypertensive rodent heart, -MHC gene expression is
significantly increased relative to normal control values. The
upregulation of
-MHC gene expression in hypertensive adult mammalian
hearts has been well documented (16, 22, 29); however, the
molecular mechanisms driving this upregulation are poorly understood.
On the basis of nuclear run-on assays, it is established that the
transcription of the
-MHC gene is significantly increased above
control level (40). Most previous studies have attempted
to understand cardiac gene transcription regulation by using transient
transfections in cultured isolated neonatal cardiac myocytes (7,
8, 11, 26, 27, 41). When neonatal cardiomyocytes in culture are
subjected to stretch or treated with various growth factors, vasoactive
substances (endothelin and ANG II), or
1-adrenergic
agonist, the cells hypertrophy and undergo molecular changes similar to
that observed in the hypertensive heart (20, 27, 32, 36).
However, this culture model of isolated cells is not able to mimic the
complex circulatory and hemodynamic effects on the intact heart.
Furthermore, the response of mature adult myocytes might differ from
that of neonatal immature myocytes, which are used in most in vitro
studies. Thus it is critical to evaluate the molecular mechanisms in
response to pathophysiological changes in vivo, at the intact organ
level. This issue is further emphasized by the fact that some
discrepancy in the results has been found between in vivo and in vitro
studies (9).
In the present study, we used the direct gene transfer approach to
study the transcriptional activity of the -MHC gene promoter in the
intact heart in vivo in the context of its regulation in abdominal
aortic constriction (AbCon)-induced overload hypertension.
-MHC
promoter fragments of various lengths linked to the firefly luciferase
(FLuc) reporter were injected into the left ventricular apex of adult
rats at an early stage of systemic hypertension (5 days after
surgically induced hypertension), and reporter expression was studied 7 days after injection. Our objective was to characterize the role of
various
-MHC promoter sequences in the regulation of
-MHC gene
transcription in the pressure-overloaded (hypertensive) rodent heart.
More specifically, we tested the full-length (3500-bp)
-MHC promoter
activity in the AbCon heart and compared its regulation with that of
the endogenous gene. We also tested the responsiveness of
different-length
-MHC promoters in the AbCon heart to define possible involvement of a specific region or elements of the promoter in the upregulation of the gene. Finally, we introduced a series of
mutations in specific regulatory elements of the promoter and tested
them for their ability to blunt the response observed in the wild-type
promoter. Our results show that some previously defined, specific key
cis-elements in the proximal promoter are involved in
maintaining basal promoter activity; however, none of the tested key
regulatory elements, individually or collectively, play a regulatory
role in mediating the increased transcriptional activity of the
-MHC
gene promoter in response to AbCon.
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MATERIALS AND METHODS |
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Animal Model and Experimental Design
All animal-related procedures described in this study were approved by our institutional animal care and use committee. Young adult female Sprague-Dawley rats (~150 g body wt; Taconic Farms, Germantown, NY) were used for all experiments. For each construct tested, 10 rats were assigned to the normal control (NC) group, while 20 rats were assigned to the AbCon group. In animals comprising the AbCon group, hypertension was induced via AbCon 5 days before plasmid injections. Rats were anesthetized with a mixture of ketamine, acepromazine, and xylazine (50, 1, and 4 mg/kg, respectively). The abdominal aorta was surgically isolated proximal to the renal arteries. A 2-0 silk suture was tied tightly around a blunt 22-gauge needle placed along the side of the aorta above both renal arteries and in proximity to the junction of the right renal artery. The needle was removed, leaving the vessel constricted. The abdomen was closed with sterile surgical sutures, and the animals were allowed to recover before they were returned to their vivarium cages. In the experiments presented here, mortality due to the AbCon procedure was ~21.5%.DNA Injection Procedure
After the rats underwent general anesthesia with ketamine, acepromazine, and xylazine (50, 1, and 4 mg/kg, respectively), the DNA injection into the myocardium was performed via a subdiaphragmatic approach similar to that described by Guzman et al. (15) and exactly as described previously (44). Mortality due to the plasmid injection did not exceed 3%.Tissue Processing
Seven days after the DNA injection, the animals were deeply sedated with a lethal dose of pentobarbital sodium (Nembutal, 100 mg/kg). Each heart was rapidly excised, and the ventricles were dissected out free of atria and major blood vessels, rinsed in cold saline, blotted dry, weighed, and cut into apex (containing the plasmid-injected area) and base portions (saved for nuclei isolation). The heart portions were then quickly frozen on dry ice and stored atBlood Pressure Measurements
To validate our experimental model in a separate experiment, we first determined that the AbCon procedure causes significant elevation of arterial pressure between 6 and 12 days after AbCon surgery, during the critical period in which the injected plasmid constructs were exposed to the myocytes' nuclear milieu (Table 1). Arterial blood pressures were measured in NC rats, as well as in AbCon rats, 6 and 12 days after they underwent the AbCon surgical procedure (n = 8/group). Animals of 140-180 g at time of surgery were anesthetized with a ketamine-acepromazine-xylazine cocktail (50, 1, and 4 mg/kg, respectively). The right carotid was isolated using blunt dissection. A polyethylene (PE-50) catheter was filled with heparinized saline (10 U/ml) and plugged at one end. The catheter was inserted and advanced into the carotid lumen. It was then secured with 2-0 silk suture, tunneled under the skin, and exteriorized at the scapular region. Animals recovered for 24 h before measurements. For measurements, the catheter was unplugged, flushed with heparinized saline, and connected to a blood pressure transducer (model TSD 104A, Biopac Systems). The pressure recordings were done via computer using Biopac Systems hardware interface and software (MP 100 system).
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Plasmid Constructs
The plasmids (The 3500-,
408-, and
215-bp
-MHC-pGL3 constructs are the same
as those used previously (44). The
299-,
171-, and
71-bp promoter constructs were generated by PCR using the
408-bp
construct as a template and Pfu high-fidelity DNA polymerase
(Stratagene). The upstream sense primers were
-MHC promoter
sequences from
299 to
280 bp for the
299-bp fragment
amplification, from
171 to
152 bp for the
171-bp fragment
amplification, and from
71 to
52 bp for the
71-bp fragment
amplification; the upstream primers were designed to contain an
NheI site at their 5' ends to allow digestion and ligation
in specific sites of the pGL3 vector. The downstream antisense primer
was the same for all amplifications, and its sequence was complementary
to that linking the
-MHC promoter at the +34 position to the pGL3
sequence, spanning 26 bp and containing the HindIII site in
its center. Subsequent to the amplifications, the PCR products were
purified by gel electrophoresis and extraction (Qiagen gel extraction
kit) and subjected to NheI-HindIII restriction digest. The digested fragments were purified by gel electrophoresis and
extraction (Qiagen) and ligated into the pGL3 basic vector at the
NheI-HindIII multicloning site. All promoter
constructs were sequenced and examined for possible unwanted mutations
due to the PCR amplification procedure.
To account for differences in DNA uptake, a fixed amount (8.03 µg) of
the -MHC-pRL plasmid, expressing the RLuc reporter gene, was
coinjected with the test
-MHC-pGL3 plasmid in all experiments, and
its activity was used to correct for variation in gene transfer efficiency. Thus
-MHC promoter activity was expressed relative to
-MHC-pRL by the FLuc-to-RLuc ratio, as described previously (44). It had been demonstrated previously by direct gene
transfer into AbCon hearts (20, 34) that the
-MHC gene
transcription remains unchanged in the AbCon vs. NC hearts. This and
the fact that the
-MHC gene is a cardiac muscle-specific gene that
is coexpressed with the
-MHC gene in the same myocytes
(37) make the
-MHC-pRL promoter construct the ideal
control reference for all direct gene transfer experiments involving
the AbCon model. The
-MHC promoter
2936 to +420 bp was a kind gift
from Dr. Eugene Morkin (University of Arizona, Tucson, AZ)
(42).
Plasmids were amplified in Escherichia coli cultures and purified by anion-exchange chromatography using disposable columns (Endofree Maxiprep, Qiagen). Plasmids were suspended in sterile PBS, and the concentration was determined by ultraviolet absorbance at 260 nm, using the conversion factor of 50 µg/ml per optical density (OD) unit. Plasmid preparations were examined by ethidium bromide staining after agarose gel electrophoresis to verify their supercoiled nature and their freedom from genomic DNA and cellular RNA.
Site-Directed Mutagenesis
The MORPH mutagenesis kit and protocol from 5primeA 408-bp
e1 mutant containing a 4-bp mutation introduced
into the promoter sequence of the
408-bp
-MHC construct within the
e1 regulatory element.
This mutation consisted of changing GGTGG to CATAT (see Table
2 for mutagenic oligonucleotide
sequence).
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A 408-bp
e2/
e3 double mutant containing two
consecutive 3-bp mutations introduced into the promoter sequence of the
408-bp
-MHC construct within the
e2 and
e3 regulatory
elements.
This mutation consisted of changing GTG to TGT in the
e2 element and
changing ACC to CGG in the
e3 element (see Table 2 for mutagenic
oligonucleotide sequence).
A 408-bp
e2/GATA/
e3 triple mutant consisting of a
4-bp mutation introduced into the promoter sequence of the
408-bp
e2/
e3 double mutant at the site of the GATA sequence
adjacent to the
e2 element.
This mutation consisted of changing GATAT to
ACTCG (see Table 2 for mutagenic oligonucleotide sequence).
A 215-bp
e3 mutant containing a 3-bp mutation introduced
into the promoter sequence of the
215-bp
-MHC construct within the
e3 regulatory element.
This mutation was identical to the
e3 mutation in the
408-bp
e2/
e3 double-mutant construct described above.
Reporter Gene Assays
Frozen cardiac tissue (~200 mg) from the apex was homogenized in 2 ml of an ice-cold lysis buffer (Promega) made up in nuclease-free water, supplemented with 5 µg/ml aprotinin, 2.5 µg/ml leupeptin, and 0.2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (protease inhibitors; Sigma Chemical), with the use of a glass homogenizer. A 0.25-ml aliquot of the total homogenate was immediately transferred to a tube containing 0.75 ml of TriReagent-LS for liquid samples (Molecular Research Center, Cincinnati, OH), rapidly mixed, and stored atMHC mRNA Analysis
The cardiac MHC mRNA profile was determined in all heart samples from each experiment to verify that the AbCon model was effective in inducing the endogenousAnalyses of MHC mRNA isoforms utilized a modification of a previously
used RT-PCR technique designed to quantitate relative amounts of MHC
mRNAs representing the various MHC isoforms ( and
), as described
in detail previously for skeletal muscle MHC isoforms (6).
RT.
For each sample, 1 µg of total RNA was reverse transcribed using the
SuperScript II RT (GIBCO BRL) and oligo(dT) primers according to the
provided protocol. At the end of the RT reaction, the tubes were heated
at 85°C for 5 min to stop the reaction and stored frozen at 80°C
until they were used in the PCR.
PCR primers and internal control fragment.
The 5'-upstream primer was designed from a highly conserved region in
all known rat MHC genes located at ~500-550 bp upstream from the
stop codon. All known seven MHC mRNA isoforms are identical in
this region spanning ~35 nt. A 20-nt oligonucleotide of the following
common sequence was chosen: 5'-AGAAGGAGCAGGACACCAGC-3'. It is the same
5' primer used to amplify skeletal MHC mRNA as described previously
(1). The 3'--MHC isoform-specific oligonucleotides used
in the PCR was the same as that used previously (6, 43). The 3'-
-MHC-specific primer used in the amplification is the same as
that used for Northern hybridization and is of the following sequence:
5'-GTGGGATAGCAACAGCGAGGC-3'.
PCRs. Each RT reaction was diluted 40-fold with nuclease-free water and mixed with an equal volume of the control fragment at the appropriate dilution (~1 amol/µl). Two microliters of this mixture were used for 25-µl PCRs, which were carried out as described previously (1, 6). PCR products were separated on a 2% agarose gel by electrophoresis, and signal quantification was done as reported previously (6, 43). This method was used to calculate the expression of each specific MHC mRNA isoform relative to the total MHC mRNA pool.
Gel Mobility Shift Assays
Nuclear extract was prepared from the base portions of NC and AbCon hearts as described earlier (17). Double-stranded oligonucleotides consisted of 20-30 bp from theStatistical Analysis
Values are means ± SE. All statistical tests were performed using Graphpad software (Prism 3.0). One-way ANOVA was used for multigroup comparisons, followed by Newman-Keuls post hoc test. A two-tailed unpaired t-test was used for two-group comparisons. P < 0.05 was taken as the level of statistical significance. ![]() |
RESULTS |
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Validation of the Hypertension Model
In the experiments comprising this project, promoter-reporter plasmid injections into the heart muscle were performed at 5 days after induction of the hypertension (via AbCon surgery), and the hearts were analyzed for reporter activity 7 days later, i.e., 12 days after induction of hypertension. Therefore, it was important to first ensure that a hypertensive state was present between 6 and 12 days after initiation of the AbCon, because this represented a critical time during which the reporter constructs were exposed to the nuclear milieu of the induced heart myocytes.Our results show that in the AbCon groups the mean blood pressure
increased by 45 and 56% after 6 and 12 days, respectively (P < 0.05), compared with the NC animals (Table 1).
Also the hearts were significantly enlarged by 26% at 6 days and by
35% at 12 days on the basis of the ventricular weight-to-body weight ratio (Table 1). Endogenous -MHC mRNA analysis shows that, after 12 days of AbCon, the
-MHC mRNA proportion was significantly increased
from 12 ± 2% in the NC hearts to 38 ± 2% in the AbCon hearts (Table 1). These findings confirm that 6-12 days after the
AbCon surgery the heart was significantly altered and underwent changes
at the molecular level that resulted in a phenotype change. Therefore,
this model was most suitable to study transcriptional events affecting
the activity of the
-MHC gene promoter.
Transcriptional Activity of the 3500-bp -MHC Promoter in the
AbCon Heart: Validation of the Gene Injection Protocol
There is strong evidence that the -MHC promoter is a good reference
promoter in interpreting the transcriptional activity of the
-MHC
promoter under normal conditions. However, when studies involve
experimental manipulations, for correct interpretation of the test
promoter activity, the control reference promoter activity (used to
normalize the response) should ideally not change in response to these
manipulations. Also, it is important to ensure that the manipulation
does not affect differentially the posttranscriptional handling of the
reporter genes (FLuc and RLuc), such as mRNA and protein synthesis and
degradation rates.
Little is known about the transcriptional activity of the -MHC
promoter in AbCon hearts, especially in the early stage of the
hypertensive response. However, Ojamaa et al. (34) and
Herzig et al. (20) found that the
-MHC promoter
activity was not altered in the AbCon model under conditions similar to
our experimental model, although the
-MHC mRNA expression was
slightly but significantly decreased. This was attributed to
posttranscriptional control of the
-MHC gene expression in the AbCon
hearts (20, 34).
Our results show that when the 3500-bp
-MHC-pGL3 and
-MHC-pRL
were cotransfected in NC and AbCon hearts, the
-MHC promoter activity relative to
(
-to-
ratio) was induced by ~385%
over the NC expression level (Fig.
1A). Analysis of the net
-MHC promoter activity in the heart of NC and AbCon rats without any
correction shows that
-MHC activity was significantly increased by
~160% (P < 0.01) in the AbCon group (Fig.
1B). Along with these changes, the net activity of the
-MHC promoter was decreased by ~33% (Fig. 1C), but
this decrease was not statistically significant (P = 0.2). We speculate that this apparent 33% decrease in the
-MHC activity is the result of a reduced gene uptake. In studying the various
-MHC promoter constructs tested in this study (total of 10 different constructs), it was found that, in all
-MHC
promoter-containing constructs, the net
-MHC activity was
consistently higher in the AbCon than in the NC hearts, while the
-MHC promoter activity, on average, was not different between NC and
AbCon hearts (Fig. 1D). For example, in these separate
experiments, a fixed amount of the
-MHC-pRL (8.03 µg/apex) was
coinjected with the various tested
-MHC promoter constructs in the
NC vs. AbCon model. When the
-MHC promoter net activity was averaged
across all 10 experiments, the
-MHC activity was similar in NC
(n = 97) and AbCon (n = 142) hearts
(Fig. 1D), thus validating the use of
-MHC promoter as a
reference control to correct for plasmid uptake in the AbCon model.
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Furthermore, the data in Fig. 1 show that the exogenous 3500-bp -MHC
promoter is active in the normal rodent heart, and this activity is
induced ~3.8-fold in response to AbCon. However, this induction in
reporter activity ratio (FLuc/RLuc) could be attributed (at least in
part) to differential effects of pressure overload on
posttranscriptional handling of the reporter genes such as mRNA and
protein stability. For example, preferentially, accumulation of FLuc
protein over RLuc protein in the AbCon hearts would result in a higher
FLuc/RLuc without necessarily a higher transcription rate of the FLuc
gene. To test the effects of AbCon on FLuc/RLuc, the promoterless pGL3
plasmid was coinjected with the
-MHC-pRL plasmid in NC and AbCon
hearts in the same way as was done for all the tested
-MHC promoter
constructs (see MATERIALS AND METHODS). Hearts injected
with the promoterless pGL3 basic plasmid have a low background level of
FLuc expression that can be accurately determined via the reporter
assay system (see MATERIALS AND METHODS). The background
activity resulting from promoterless plasmid injection was consistently
>20-fold higher that of uninjected tissue extract activity. Thus the
promoterless pGL3 plasmid injection is a good approach to test the
effects of the AbCon model on FLuc stability without the confounding
effects from transcription. The results of these injections are
reported in Fig. 2. The data show that the net activity levels of FLuc (Fig. 2A) or RLuc (Fig.
2B) were unchanged in response to AbCon. More importantly,
RLuc/FLuc was not different in the AbCon hearts (Fig. 2C),
although the endogenous
-MHC mRNA levels were induced to 36 ± 3%, and heart weight-to-body weight ratio increased by 34 ± 2%
(Table 3). These results indicate that
any induction in this FLuc/RLuc (as observed in Fig. 1A) in
the AbCon hearts must indeed result from a change in the
-MHC promoter activity upstream to the FLuc gene.
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Thus we have confirmed that the changes in -MHC gene expression due
to AbCon are mainly regulated at the transcriptional level and that a
deletion analysis of the promoter would enable us to delineate
important sequences of the promoter that are responsible for
regulating the hypertension-induced increase in
-MHC gene transcription.
Deletion Analysis of the -MHC Promoter in NC and AbCon
Hearts
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When the same constructs were studied in AbCon hearts, all the tested
deletions, including the short 71-bp construct, of the
-MHC
promoter showed significant upregulation of the
-to-
reporter
ratio. The 3500-bp full-length promoter showed maximal responsiveness:
an approximately fourfold increase in the
-to-
reporter ratio
compared with the NC hearts (Fig. 3C). The
408- and
299-bp
-to-
reporter ratios were induced to a similar level by
155 and 157%, respectively, in response to AbCon, but this responsiveness was 60% lower than that of the
3500-bp promoter (P < 0.05; Fig. 3C). The
215- and
171-bp promoter
activities were induced by 67 and 87%, respectively (Fig.
3C). Interestingly, although the
71-bp promoter construct
had lost almost all of its activity, upregulation of reporter gene
activity in hypertension was the same as in the
171-bp construct
(87%), indicating that the promoter sequence located between
171 and
71 bp is crucial for basal expression of the gene but is not involved
in mediating the hypertension response. Unlike the
71-bp
-MHC
promoter construct, the promoterless construct (pGL3 basic) reporter
activity did not show responsiveness to hypertension (Figs. 2 and
3C), suggesting that the upregulation observed in the
71-bp
-MHC promoter is specific to the
-promoter and not a
generalized response to AbCon. This strongly indicates that a
regulatory element that is involved in hypertension regulation of the
gene is located between the TSS and position
71 of the
-MHC promoter.
These results suggest that full regulation of -MHC gene expression
in the AbCon model requires the full-length promoter (i.e.,
3500 to
+34 bp). However, because regulation is also significant in the
shortest promoter studied (
71 bp), which is lacking all the known
positive regulatory elements, the hypertension response appears to be
regulated through several elements located in different regions of the
promoter. The level of induction of the
299-bp promoter fragment was
identical to that of the
408-bp fragment, which suggests that the
e1 repressor element or other promoter sequences located between
408 and
299 bp (Fig. 3) are not critically involved with the
upregulation of the
-MHC gene expression in the AbCon model. The
significant decrease in responsiveness when the
299-bp
-MHC
response is compared with the
215-bp
-MHC response (Fig.
3C) suggests that some sequences located between
299 and
215 bp relative to TSS are responsible for the
-MHC upregulation
in response to hypertension. In this region, three regulatory elements
are defined: the
e2 element, GATA (AT rich), and C-rich regions
(Fig. 3A). As more sequences were removed between
215 and
71 bp, including the loss of the
e3 element (41) and
a nuclear factor of activated T cells (NF-AT)-like binding site
(35), no further reduction in the responsiveness to AbCon was observed (Fig. 3C), indicating that no hypertension
response elements are located between
215 and
71 bp. In conclusion,
at least three regions of the
-MHC promoter appear to be involved in
hypertension regulation of the gene: 1) upstream sequences between
408 and
3500 bp, 2) sequences between
299 and
215 bp, and 3) sequences between
71 bp and the TSS.
It is important to note that the differences in responsiveness of these
promoter fragments to AbCon cannot be attributed to differences in the
endogenous response. On the basis of ventricular weight-to-body weight
ratios and endogenous -MHC mRNA expression (Table 3), the AbCon
response was relatively homogeneous.
Mutation Analysis of the 408-bp -MHC Promoter in NC and
AbCon Hearts
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Simultaneous mutations of the e2 and
e3 elements on the 408-bp
-MHC promoter did not alter the degree of responsiveness to AbCon
compared with the wild-type mutation (Fig. 4B); however, the
same mutations reduced the promoter activity in NC hearts by 39% (Fig.
4A), indicating that the
e2 and
e3 elements are positive regulatory elements but are not involved in the
transcriptional regulation in the AbCon response in the heart.
Furthermore, mutation of a GATA-like sequence, in addition to
e2 and
e3 mutations on the 408-bp
-MHC promoter, had no effect on NC
expression or responsiveness to AbCon (Fig. 4A). This result
raises a question concerning the importance of this GATA-like site in
regulating
-MHC promoter activity. At the same time, these results
confirm in the in vivo model the role of
e1,
e2, and
e3
regulatory elements in maintaining the normal expression of the
-MHC
gene but do not support their involvement in the upregulation
response to AbCon when studied in the 408-bp promoter fragment. In the AbCon groups described above, the endogenous response of the
heart to AbCon was relatively homogeneous in all AbCon groups that were studied. There was no significant difference in the AbCon-induced increase in ventricle weight-to body weight ratio or in the increased levels of the endogenous
-MHC mRNA expression among the various AbCon groups (Table 4). Thus any
difference in the reporter gene expression cannot be attributed to a
lack of homogeneity in the endogenous response to AbCon among the
various groups.
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Effect of a e3 Mutation on the 215-bp
-MHC Promoter: Activity
in NC Hearts and Responsiveness to AbCon
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Again the heart relative mass (ventricle weight-to- body weight ratio)
and percent -MHC mRNA expression were not different among the NC
groups or among the AbCon groups. A consistent increase was observed in
both AbCon groups relative to their corresponding controls (Table
5).
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Nuclear Extract Interaction With Specific Cis-Elements on the
-MHC Promoter
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In addition to e2, GATA, and
e3, we also examined the binding to
the
e1 repressor element, as well as to the TATA box, and these
results are reported in Fig. 7. NC and
AbCon heart nuclear extracts formed shifted complexes on the gel. The
specific binding to the
e1 sense strand increased by ~26% under
AbCon conditions (P < 0.05).
e1 is a repressor
element, as defined previously (7, 11), which is confirmed
by our data showing that the in vivo promoter activity increased in the
NC heart when this
e1 element was mutated in the
408-bp
-MHC
promoter construct (Fig. 4). When this element was deleted, as in the
299-bp
-MHC promoter (Fig. 3), or mutated, as in the
408-bp
-MHC promoter (Fig. 4), the responsiveness to AbCon was still
similar to that of the wild-type promoter, thus eliminating a potential
role played by
e1 in the AbCon induction of the promoter activity.
If
e1 were a player in the AbCon-induced
-MHC promoter
upregulation, one might expect to find a decreased repressor
activity/nuclear extract-
e1 interaction in AbCon hearts, but that
was not the case. In contrast, a trend for the opposite was observed,
i.e., increased
e1 binding (Fig. 7). The significance of this
observation remains unclear; thus more research is needed in this area.
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Because promoter activity of the shortest tested fragment, 71 bp, was
also upregulated in response to AbCon, one could conclude that the
basal promoter activity is induced. The basal promoter consists of
downstream sequences located within 100 bp at the 3' end of the
promoter, and these include the TATA box. Several nuclear proteins
interact at the basal promoter region to form a multimeric complex
required for transcription initiation. To establish whether AbCon
hearts are associated with increased binding activity to the TATA
element in the basal
-MHC promoter, a gel mobility shift assay was
used to test the interaction between the
-MHC TATA element and
nuclear extract from normal vs. AbCon hearts. Our results reported in
Fig. 7 show no significant difference between NC and AbCon hearts'
binding activities to TATA. Thus increased TATA binding protein, in and
of itself, is not an explanation for the increased promoter activity in
the AbCon hearts. One possible explanation is that other factors in the
basal promoter machinery are activated, or other regulatory
transcription factors are involved. Further deletions and studies of
the activity of shorter fragments are required to characterize the
-MHC promoter response to AbCon (see below).
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DISCUSSION |
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The heart responds to systemic hypertension by altering the
expression of various genes that are important for economizing the
contractile properties of the heart. The -MHC gene is known to be
upregulated in the hypertensive rodent heart. However, the mechanism by
which this upregulation in gene expression is mediated is not well
understood. Previously, tissue culture studies have attempted to
delineate regions on the
-MHC promoter that might be involved in
mediating the hypertension response of this gene. Because of the
complexity of the stimulus (mechanical, hormonal, neuronal), it is very
difficult to create a useful tissue culture model of hypertension.
Furthermore, it is important to study gene regulation in vivo under
physiological conditions, because it has been shown that the results
from in vitro studies of the gene can be in discordance with the
results from in vivo studies. For example, Edwards and Ghaleh
(9) tested
-MHC promoter deletion constructs containing
the rat or human
e2 element under in vitro and in vivo conditions.
When promoter/reporter constructs were transfected into cultured fetal
rat cardiomyocytes, the human
-reporter was expressed more
than threefold above the equivalent rat construct. However, when these
same
-MHC promoter constructs were injected into adult rat
hearts (in vivo), the levels of reporter expression were similar
for the human and the rat constructs (9).
Generally, two approaches have been used to study -MHC gene
expression in vivo (in the intact animal): 1) the use of
transgenic mice and 2) direct gene injection into the muscle
tissue. In a previous study, we successfully used a direct gene
injection approach that allows gene transfection of the myocardial
cells in vivo in the intact heart (44). In the present
study, this method was applied to study the regulation of the
transcriptional activity of the
-MHC promoter in response to
hypertension in vivo. We prefer this method of studying the promoter
over transgenics, because it is less costly and time consuming. The
activity of the test promoter was normalized to that of a reference
promoter consisting of the
-MHC promoter linked to the RLuc reporter
gene. The effectiveness of the AbCon procedure in inducing hypertension was proven with significant increases in mean blood pressures and
relative ventricular weights (Fig. 1, Table 1). The
-MHC mRNA
expression increased significantly as a result of the AbCon procedure
(Table 1), and this was associated with a parallel increase in the
transcriptional activity of the longest (
3500-bp)
-MHC promoter
(Fig. 1). These results make our model suitable to study
transcriptional regulation of the
-MHC promoter in the hypertensive
heart. In contrast, the
-MHC expression does not change appreciably
under the same conditions when examined at the transcriptional level
(Fig. 1D) (20, 34) or the mRNA level (23). The
-MHC promoter, therefore, presents a suitable
reference to correct for variability in plasmid transfection efficiency associated with the direct gene injection experiments. In the present
report, the
-MHC promoter activity is expressed as the
-to-
ratio of reporter gene expression in NC and hypertensive rodent hearts.
Deletion analysis of the -MHC promoter revealed that upstream
sequences are necessary for full regulation of the promoter in
hypertension (Fig. 3). This is consistent with our previous reports on
the heart (44) and on regulation of the gene in skeletal muscle (12). Sequence analysis of these upstream sequences
showed several sites with a high degree of identity to known regulatory elements. Of particular interest are MCAT elements (10,
26), E boxes (3), and NF-AT-like binding sites
(35), all of which are present in the upstream sequences
of the
-MHC promoter. Although these sites would be interesting
targets of deletion and mutation studies, the focus of this report was
on the proximal promoter region of the
-MHC promoter for the
following reasons: 1) This proximal promoter region of
~400 bp does confer responsiveness of the gene to hypertension and
must, therefore, contain at least one hypertension response element.
2) Several regulatory sites have been identified in this
region, and some have even been proposed as hypertension response
elements (19, 26, 27). These regulatory elements are
specific targets for mutation analysis. 3) The proximal promoter region enables us to compare results obtained with in vitro
tissue culture studies with our in vivo model.
Tissue culture studies have identified a number of regulatory elements
in the proximal promoter region (from the TSS to 400 bp):
e2,
e3, and C-rich are positive regulators of
-MHC gene expression
that have been recognized in cardiac and skeletal muscle cell culture
studies (41). In addition, the
e1 element and at least
one thyroid response element (TRE) have been identified as negative
regulators of
-MHC gene expression (7, 8, 44). The
results of this study confirm other findings that
e2 and
e3 are
positive regulatory elements (19), whereas
e1 is a repressor element (7) regulating the expression of the
-MHC gene in the normal intact heart.
Of particular interest in the context of hypertension regulation are
the e2 and
e3 elements, since they are positive regulators of
-MHC gene expression. The
e2 element contains an MCAT motif that
serves as a binding site for the transcriptional enhancer factor 1 (TEF-1), also referred to as the MCAT-binding protein (MBP) (10,
26). The
e3 element contains another MCAT motif that is also
a recognition site for the MBP (26). Mutation of this
element has indicated that it is essential for
-MHC gene activation
by
-adrenergic stimulation or protein kinase C activation in vitro
(27). The C-rich element (CCAC box) is a potential binding
site for the ubiquitous transcription factor SP-1. This factor has been
shown to function in cooperation with the
e2 element in the
activation of
-MHC gene transcription in cardiomyocytes in vitro
(41).
Recently, a GATA element that is located directly adjacent to the e2
element has been implicated as an important regulator of
-MHC
transcription or even as the hypertension response element of the gene
in vivo (19). These authors propose that a
303- to
197-bp region of the promoter is responsible for hypertension activation (19). Hypertension induction of the gene was
completely lost in the shortest (
203-bp) promoter fragment tested.
Several known promoter elements are located in this 106-bp region:
e2 (
285 to
269 bp), part of
e3 (
210 to
188 bp), and a
GATA element and the C-rich element (CCAC box,
250 to
230 bp).
Hasegawa et al. (19) suggested that simultaneous mutation
of the
e2 and
e3 elements did not change aortic
constriction-stimulated transcription; however, mutation of the GATA
element, which is adjacent to the
e2 element, markedly decreased
aortic constriction-stimulated transcription. Therefore, it was
concluded that the GATA element is a hypertension response element
directly involved in upregulating the activity of the
-MHC gene
promoter (19).
The results of this study also indicate that a hypertension response
element is located between 299 and
215 bp, because promoter
activity induction in hypertensive hearts was significantly lower in
the
215- than in the
299-bp promoter segment (Fig. 3C).
However, in our hands, neither the
e2/
e3
408-bp double mutant
nor the
e2/
e3/GATA
408-bp triple mutant had a significant effect on blunting the hypertension-induced transcriptional regulation of the gene (Fig. 4). The reason for this discrepancy is not apparent, but it could be due to the difference in the normalization of the test
promoter: Hasegawa et al. (19) used a viral promoter as a
reference promoter to correct for variability in transfection efficiency, whereas in this study the
-MHC promoter was used. The
only other known regulatory element in the region between
299 and
215 bp is the C-rich element (CCAC box). Preliminary results suggest
that this element might indeed be involved in regulating the response
of the gene to hypertension (unpublished observation). Our results also
indicate that at least one other hypertension response element must be
located within 71 bp from the TSS, since the shortest tested promoter
fragment (
71 bp) still showed a significant response to hypertension,
while the promoterless construct had no response to hypertension (Fig.
3C). These results are in contrast to the report of Hasegawa
et al. on the lack of responsiveness of the rat
203-bp
-MHC
promoter fragment. The short
71-bp promoter construct was
particularly interesting, because although almost all (~97%) of the
reporter activity seen in the next-larger construct (
171 bp) was lost under NC conditions, the responsiveness to hypertension was identical to that of the
171-bp promoter construct (Fig. 3). This indicates that the regulatory elements that are located between
71 and
171 bp
on the promoter are vital for the general transcription of the gene but
not for directing the transcriptional regulation in response to hypertension.
In conclusion, the results of the experiments in this report indicate
that three separate regions on the -MHC promoter are involved in the
induction of the gene in response to hypertension: 1) a
distal region located between
408 and
3500 bp (possibly in
conjunction with the previously identified upstream enhancer) (44), 2) a proximal region located between
299 and
215 bp (likely involving the C-rich element), and
3) a basal region within 71 bp upstream of the TSS.
Therefore, future research needs to further characterize these
responsive regions of the promoter to more fully delineate
-MHC
transcriptional regulation in response to pressure overload.
![]() |
ACKNOWLEDGEMENTS |
---|
The authors thank Jason Chang and Christopher Ma for technical assistance.
![]() |
FOOTNOTES |
---|
This work was supported by National Heart, Lung, and Blood Institute Grant HL-38819 (to K. M. Baldwin).
Address for reprint requests and other correspondence: K. M. Baldwin, Dept. of Physiology and Biophysics, University of California, Irvine, Irvine, CA 92697 (E-mail: kmbaldwi{at}uci.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 17 October 2000; accepted in final form 8 December 2000.
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