In vivo regulation of the beta -myosin heavy chain gene in hypertensive rodent heart

Carola E. Wright, P. W. Bodell, F. Haddad, A. X. Qin, and K. M. Baldwin

Department of Physiology and Biophysics, University of California, Irvine, California 92697


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The main goal of this study was to examine the transcriptional activity of different-length beta -myosin heavy chain (beta -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 beta -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 beta e1, beta e2, GATA, and beta 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 beta -MHC gene. Collectively, these results indicate that three separate regions on the beta -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 beta -MHC transcriptional regulation in response to pressure overload.

hypertension; transcription; dual luciferase; in vivo gene transfer


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ADULT MAMMALIAN CARDIAC MUSCLE expresses two genes encoding for myosin heavy chains (MHCs), which have been designated alpha - and beta -MHC (28). The alpha - and beta -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 alpha -MHC is expressed only in the heart, beta -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 alpha -MHC isoform is characterized by a higher ATPase activity and faster shortening speed than the beta -MHC isoform (2, 21). Thus hearts rich in the alpha -isoform have a high intrinsic contractility, whereas those rich in the beta -isoform have a lower contractility but a higher economy of tension development (2). In the adult rodent, the alpha -MHC is the predominant isoform expressed in the ventricles, accounting for ~85-90% of the total MHC protein pool, whereas the beta -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, beta -MHC gene expression is significantly increased relative to normal control values. The upregulation of beta -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 beta -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 alpha 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 beta -MHC gene promoter in the intact heart in vivo in the context of its regulation in abdominal aortic constriction (AbCon)-induced overload hypertension. beta -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 beta -MHC promoter sequences in the regulation of beta -MHC gene transcription in the pressure-overloaded (hypertensive) rodent heart. More specifically, we tested the full-length (3500-bp) beta -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 beta -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 beta -MHC gene promoter in response to AbCon.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 at -80°C until subsequent processing.

Blood 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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Table 1.   Mean blood pressure measurements, ventricular weight-to-body weight ratios, and endogenous beta -MHC mRNA expression in NC vs. AbCon hearts after 6 and 12 days

Plasmid Constructs

The plasmids (-3300 to +34)- and (-215 to +34 bp)-beta -MHC chloramphenicol acetyltransferase (CAT) were a kind gift from Dr. P. C. Simpson (University of California, San Francisco) and contained a rat beta -MHC genomic fragment fused to the CAT reporter sequence in pUC9 (27). An additional construct, containing a -3500- to +462-bp [from the transcription start site (TSS)] beta -MHC genomic sequence fused to an FLuc reporter plasmid was kindly provided by Dr. Kaie Ojamaa (33). Promoterless reporter vectors pGL3 basic and pRL null, containing the FLuc and the Renilla luciferase (RLuc) reporter genes, respectively, were purchased from Promega (Madison, WI).

The -3500-, -408-, and -215-bp beta -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 beta -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 beta -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 alpha -MHC-pRL plasmid, expressing the RLuc reporter gene, was coinjected with the test beta -MHC-pGL3 plasmid in all experiments, and its activity was used to correct for variation in gene transfer efficiency. Thus beta -MHC promoter activity was expressed relative to alpha -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 alpha -MHC gene transcription remains unchanged in the AbCon vs. NC hearts. This and the fact that the alpha -MHC gene is a cardiac muscle-specific gene that is coexpressed with the beta -MHC gene in the same myocytes (37) make the alpha -MHC-pRL promoter construct the ideal control reference for all direct gene transfer experiments involving the AbCon model. The alpha -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 5prime right-arrow 3prime, Inc. (Boulder, CO) were used for all mutagenesis reactions. Three or four bases were mutated in the target promoter sequence, and they were designed to disrupt specific transcription factor-binding sites.

A -408-bp beta e1 mutant containing a 4-bp mutation introduced into the promoter sequence of the -408-bp beta -MHC construct within the beta e1 regulatory element. This mutation consisted of changing GGTGG to CATAT (see Table 2 for mutagenic oligonucleotide sequence).

                              
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Table 2.   Oligonucleotides used for the different mutations and those used as probes or competitors in the gel mobility shift assays

A -408-bp beta e2/beta e3 double mutant containing two consecutive 3-bp mutations introduced into the promoter sequence of the -408-bp beta -MHC construct within the beta e2 and beta e3 regulatory elements. This mutation consisted of changing GTG to TGT in the beta e2 element and changing ACC to CGG in the beta e3 element (see Table 2 for mutagenic oligonucleotide sequence).

A -408-bp beta e2/GATA/beta e3 triple mutant consisting of a 4-bp mutation introduced into the promoter sequence of the -408-bp beta e2/beta e3 double mutant at the site of the GATA sequence adjacent to the beta e2 element. This mutation consisted of changing GATAT to ACTCG (see Table 2 for mutagenic oligonucleotide sequence).

A -215-bp beta e3 mutant containing a 3-bp mutation introduced into the promoter sequence of the -215-bp beta -MHC construct within the beta e3 regulatory element. This mutation was identical to the beta e3 mutation in the -408-bp beta e2/beta e3 double-mutant construct described above.

Each mutation was verified by sequencing using an automated sequencer (Applied Biosystems). The disruptive effect of each mutation on transcription factor binding was verified by us and others using gel mobility shift assays (26, 41); furthermore, the mutated sequence was checked to verify that we did not generate a potential binding site for known transcription factors (Mat Inspector version 2.2 at web site: http://transfac.gbf.de/cgi-bin/matSearch/matsearch.pl).

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 at -80°C for subsequent RNA extraction. The remainder of the homogenate was centrifuged at 4°C at 10,000 g for 10 min. The supernatant was separated and kept on ice until assayed for luciferase activities. FLuc and RLuc activities were measured from the same extract (5 µl) in a single tube using Promega's dual-luciferase protocol, as described previously (44).

MHC 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 endogenous beta -MHC gene expression. Approximately 5% of the AbCon hearts did not exhibit a significant increase in beta -MHC mRNA relative to NC, and these were excluded from the final analysis with regard to reporter gene expression. Total RNA was extracted from an aliquot of the total homogenate used for reporter gene assay using the TriReagent-LS for liquid samples, according to the supplied protocol (Molecular Research Center). Total RNA was precipitated from the aqueous phase with isopropanol, and after it was washed with ethanol, it was dried and suspended in a small volume (40 µl) of nuclease-free water. The RNA concentration was determined by OD at 260 nm (OD260 = 40 µg/ml). The RNA samples were stored frozen at -80°C until they were subsequently analyzed for MHC mRNA expression by RT-PCR technology (see below).

Analyses 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 (alpha  and beta ), 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'-beta -MHC isoform-specific oligonucleotides used in the PCR was the same as that used previously (6, 43). The 3'-alpha -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'.

The internal control fragment also was a modified version of that used for skeletal MHC isoforms. The alpha -MHC-specific antisense primer was linked in tandem to the beta  (type I)-MHC-specific primer at the 3' end, so that amplification by PCR is possible by using sets of primers (common/beta or common/alpha ) yielding PCR products of 224 or 250 bp, respectively.

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 the beta -MHC gene promoter spanning specific regulatory elements. The sequences of sense strands are reported in Table 2. For the binding reaction, nuclear extract equivalent to 1 µg of nuclear DNA (17) was preincubated for 10 min on ice with 2 µg of nonspecific homologous DNA [poly(dI-dC)] in a buffer containing 80 mM KCl, 20 mM HEPES, pH 7.9, 10% glycerol, and 1% BSA. At the end of this preincubation, ~50,000 cpm of 5'-end-labeled double-stranded oligonucleotides were added, and the reactions (20 µl total volume) were incubated at room temperature for 20 min; then reactions were stopped by addition of 2 µl of loading buffer (10% Ficoll, 0.01% xylene cyanol, and 0.01% bromphenol blue). Immediately, the reactions were loaded on a 6% polyacrylamide gel and electrophoresed at 200 V and 20 mA for 3 h under nondenaturing conditions with 0.5× Tris base-boric acid acid-EDTA as the running buffer. At the end of electrophoresis, the gels were dried and exposed to a PhosphorImager storage screen for 12-24 h; then the gel images were obtained by laser scanning of the storage screen using a PhosphorImager densitometer (Molecular Dynamics). For competition experiments, 100- to 300-fold molar excess of unlabeled probes was included in the preincubation described above, before addition of the labeled probe. Shifted bands intensity was determined using the Image Quant 4.0 software package (Molecular Dynamics).

Statistical 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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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -MHC mRNA analysis shows that, after 12 days of AbCon, the beta -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 beta -MHC gene promoter.

Transcriptional Activity of the 3500-bp beta -MHC Promoter in the AbCon Heart: Validation of the Gene Injection Protocol

In the present study, the direct gene transfer approach was used to test the transcriptional activity of the beta -MHC promoter linked to an FLuc reporter under NC and AbCon conditions. In a previous study, we used the alpha -MHC-pRL consisting of the alpha -MHC promoter linked to the RLuc reporter gene to correct for cardiac gene transfer efficiency (44). This alpha -MHC-pRL makes an ideal reference gene under normal conditions for the following reasons: 1) the alpha -MHC promoter gene is naturally active in cardiac myocytes, along with the beta -MHC gene test promoter, and unlike strong viral promoters [simian virus-40 (SV40), rous sarcoma virus (RSV), and cytomegalovirus (CMV)], competition is less likely when both genes are used moderately in equimolar amounts; 2) the test and reference promoters are cardiac specific; thus any possibility of confounding expression in cardiac fibroblasts or other nonmyocardiac cells is unlikely; and 3) the FLuc and RLuc reporter expression can be assayed with similar detection sensitivity and range (Promega dual-luciferase assay protocol).

There is strong evidence that the alpha -MHC promoter is a good reference promoter in interpreting the transcriptional activity of the beta -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 alpha -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 alpha -MHC promoter activity was not altered in the AbCon model under conditions similar to our experimental model, although the alpha -MHC mRNA expression was slightly but significantly decreased. This was attributed to posttranscriptional control of the alpha -MHC gene expression in the AbCon hearts (20, 34).

Our results show that when the -3500-bp beta -MHC-pGL3 and alpha -MHC-pRL were cotransfected in NC and AbCon hearts, the beta -MHC promoter activity relative to alpha  (beta -to-alpha ratio) was induced by ~385% over the NC expression level (Fig. 1A). Analysis of the net beta -MHC promoter activity in the heart of NC and AbCon rats without any correction shows that beta -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 alpha -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 alpha -MHC activity is the result of a reduced gene uptake. In studying the various beta -MHC promoter constructs tested in this study (total of 10 different constructs), it was found that, in all beta -MHC promoter-containing constructs, the net beta -MHC activity was consistently higher in the AbCon than in the NC hearts, while the alpha -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 alpha -MHC-pRL (8.03 µg/apex) was coinjected with the various tested beta -MHC promoter constructs in the NC vs. AbCon model. When the alpha -MHC promoter net activity was averaged across all 10 experiments, the alpha -MHC activity was similar in NC (n = 97) and AbCon (n = 142) hearts (Fig. 1D), thus validating the use of alpha -MHC promoter as a reference control to correct for plasmid uptake in the AbCon model.


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Fig. 1.   Promoter activity of 3500-bp beta -myosin heavy chain (MHC) and alpha -MHC in normal control (NC) hearts and hearts with abdominal aortic constriction (AbCon) 7 days after plasmid injection and 12 days after AbCon. A: 3500-bp beta -MHC promoter activity as normalized to alpha -MHC and expressed as beta -MHC firefly luciferase (FLuc)-to-alpha -MHC-pRL ratio in NC and AbCon hearts. B: beta -MHC promoter activity expressed as net FLuc activity in relative light units (RLU; i.e., the difference between the actual sample and the uninjected heart sample). C: alpha -MHC promoter Renilla luciferase (RLuc) activity expressed as net RLU. D: average of pooled alpha -MHC activity in a total of 10 separate experiments done in this study, which include the deletion and mutational analysis of the beta -MHC promoter, results of which are reported in Figs. 2-4 (n = 97 for NC and 142 for AbCon). Injected plasmid mixtures consisted of 10 µg of the 3500-bp beta -MHC promoter-pGL3 and 8.03 µg of the alpha -MHC promoter-pRL (equimolar to 10 µg of 3500-bp beta -MHC-pGL3) dissolved into 40 µl of sterile PBS. Values are means ± SE; n = 10 NC and 14 AbCon. NS, not significant. *P < 0.05, AbCon vs. NC. RLU in 5 µl of tissue extract was determined using a luminometer (Monolight 2010C) and integrated over a 10-s interval.

Furthermore, the data in Fig. 1 show that the exogenous 3500-bp beta -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 alpha -MHC-pRL plasmid in NC and AbCon hearts in the same way as was done for all the tested beta -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 beta -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 beta -MHC promoter activity upstream to the FLuc gene.


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Fig. 2.   Luciferase activities in NC and AbCon hearts 7 days after promoterless pGL3 basic plasmid and alpha -MHC-pRL coinjection and 12 days after AbCon. A: FLuc activity in RLU. Activity in 5 µl of tissue extract was determined using a luminometer (Monolight 2010C) and integrated over 10-s intervals. B: RLuc activity in RLU. For FLuc, activity in the same 5 µl of tissue extract was determined using the dual-luciferase assay reagents (Promega) and a Monolight 1010C luminometer. C: FLuc-to-RLuc ratio in NC and AbCon hearts. RLU reflects the difference between the actual sample and the uninjected heart sample. Injected plasmid mixtures consisted of 5.08 µg of promoterless (basic) pGL3 and 8.03 µg of the alpha -MHC promoter-pRL (both are equimolar to 10 µg of 3500-bp beta -MHC-pGL3) dissolved into 40 µl of sterile PBS. Values are means ± SE; n = 10 NC and 14 AbCon.


                              
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Table 3.   Relative increase in ventricular weight-to-body weight ratios and percent beta -MHC mRNA expression in AbCon groups in which the specified beta -MHC promoters were studied

Thus we have confirmed that the changes in beta -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 beta -MHC gene transcription.

Deletion Analysis of the beta -MHC Promoter in NC and AbCon Hearts

In addition to the full-length beta -MHC promoter, five other shorter promoter fragments (-408, -299, -215, -171, and -71 bp; Fig. 3A) were tested for their ability to induce reporter gene expression in response to AbCon. Furthermore, a promoterless reporter vector (pGL3 basic) was also tested in NC and AbCon hearts. Reporter gene activities in NC hearts for these tested promoters are shown in Fig. 3B. Data analysis showed that these shorter promoters have reduced NC activity (Fig. 3B) compared with the full-length (-3500-bp) promoter, confirming the existence of the distal enhancer in the upstream region of the promoter (44). Activities of the -408-, -299-, -215-, and -171-bp constructs were reduced by ~30-70% compared with activity of the -3500-bp promoter. The -71-bp promoter construct had the most drastic drop in activity, an additional 97% decrease compared with the -171-bp construct, which brought its activity to the level of the promoterless construct, indicating that important regulatory elements had been deleted in the segment between -171 and -71 bp.


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Fig. 3.   Deletion analysis of the beta -MHC-pGL3 promoter-reporter construct: responsiveness to AbCon. A: schematic diagram of the different-length beta -MHC promoters tested in NC vs. AbCon hearts. The enhancer region between -2900 and -3500 bp of the promoter, as defined in previous experiments (44), is depicted. Also shown are several cis-regulatory elements on the 408-bp promoter based on previous work: beta e1, beta e2, beta e3, C-rich (41), TRE/E box (8), GATA (19), and nuclear factor of activated T cells (NF-AT)-like binding site based on sequence comparison to NF-AT-binding site consensus (35). All these promoters extend to +34 bp 3' to the transcription start site (TSS) of the gene and are inserted in the multicloning site of the pGL3 vector (Promega) just upstream to the FLuc reporter gene. B: activities of beta -MHC promoter deletions in NC hearts; activities are normalized to the coinjected alpha -MHC-pRL activity and expressed as FLuc-to-RLuc ratio. The -3500 construct has the highest activity, and the -408-, -299-, and -215-bp constructs have similar activities in NC hearts but at a lower level than the -3500-bp construct. The -171-bp fragment shows an overall lower level of activity (P < 0.05) than the -408-, -299-, and -215-bp fragments. The -71-bp and the promoterless (0) constructs have similar activities, i.e., 0.0026 ± 0.0002 and 0.0027 ± 0.0002, respectively, which are ~3% of the -171-bp construct activity. C: fold increase of the beta -MHC promoter activity (beta -to-alpha ratio) in AbCon hearts relative to NC hearts. Data show the response for all the tested deletions and the promoterless (0) construct. Each bar is the difference in activity between AbCon and NC normalized to NC [(AbCon - NC)/NC]. Different-length beta -MHC promoter constructs were injected in equal molar amount to 10 µg of 3500-bp beta -MHC pGL3. The alpha -MHC-pRL was always injected at 8.03 µg. n = 10 for each NC group and as reported in Table 3 for the AbCon groups. *P < 0.05 vs. all others; **P < 0.05 vs. all others but not 0 vs. 71; #P < 0.05 vs. 408 and 299.

When the same constructs were studied in AbCon hearts, all the tested deletions, including the short -71-bp construct, of the beta -MHC promoter showed significant upregulation of the beta -to-alpha reporter ratio. The 3500-bp full-length promoter showed maximal responsiveness: an approximately fourfold increase in the beta -to-alpha reporter ratio compared with the NC hearts (Fig. 3C). The -408- and -299-bp beta -to-alpha 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 beta -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 beta -MHC promoter is specific to the beta -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 beta -MHC promoter.

These results suggest that full regulation of beta -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 beta e1 repressor element or other promoter sequences located between -408 and -299 bp (Fig. 3) are not critically involved with the upregulation of the beta -MHC gene expression in the AbCon model. The significant decrease in responsiveness when the -299-bp beta -MHC response is compared with the -215-bp beta -MHC response (Fig. 3C) suggests that some sequences located between -299 and -215 bp relative to TSS are responsible for the beta -MHC upregulation in response to hypertension. In this region, three regulatory elements are defined: the beta 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 beta 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 beta -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 beta -MHC mRNA expression (Table 3), the AbCon response was relatively homogeneous.

Mutation Analysis of the 408-bp beta -MHC Promoter in NC and AbCon Hearts

Mutation of the beta e1 repressor element on the 408-bp beta -MHC promoter construct was associated with a 111% increase in beta -MHC promoter activity in the NC hearts (Fig. 4A); however, this mutation had no effect on activation of the promoter in the AbCon heart (Fig. 4B). This indicates that the beta e1 element is a repressor element that is not involved in regulating transcriptional activity in the AbCon response of the heart.


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Fig. 4.   Mutation analysis of the 408-bp beta -MHC promoter in NC hearts (A) and responsiveness to AbCon (B). A: promoter activity (beta -to-alpha ratios) of the 408-bp wild-type (WT) and mutated constructs in NC hearts. Note the significant increase in beta -MHC promoter activity when the beta e1 repressor element was mutated. Simultaneous mutations of the beta e2 and beta e3 elements significantly reduced the promoter activity relative to WT activity in NC hearts; however, the GATA mutation in addition to the mutation of the beta e2-beta e3 elements had no further effect on the promoter activity. B: fold increase of the promoter activity in the AbCon hearts: effects of specific mutations on the 408-bp beta -MHC promoter responsiveness to AbCon. There was no significant difference in the increase of ventricle-to-body weight ratio or in the increased levels of the endogenous beta -MHC mRNA expression in the AbCon hearts (Table 4). Values are means ± SE; n = 10 for each NC group and as reported in Table 4 for the AbCon groups. *P < 0.05 vs. WT.

Simultaneous mutations of the beta e2 and beta e3 elements on the 408-bp beta -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 beta e2 and beta 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 beta e2 and beta e3 mutations on the 408-bp beta -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 beta -MHC promoter activity. At the same time, these results confirm in the in vivo model the role of beta e1, beta e2, and beta e3 regulatory elements in maintaining the normal expression of the beta -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 beta -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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Table 4.   Relative increase in ventricular weight-to-body weight ratios and percent beta -MHC mRNA expression in AbCon groups after injection with the specified 408-bp beta -MHC-pGL3 promoter construct

Effect of a beta e3 Mutation on the 215-bp beta -MHC Promoter: Activity in NC Hearts and Responsiveness to AbCon

Mutation of the beta e3 element within the -215-bp promoter fragment was associated with a 41% decrease in beta -MHC promoter activity relative to the wild-type mutation in the NC heart (Fig. 5A). However, the responsiveness to AbCon of this mutated promoter fragment was not reduced compared with the level of gene expression of the wild-type mutation (Fig. 5B). Thus these results confirm the role of the beta e3 element as a positive regulatory element in the normal expression of the beta -MHC gene but do not support its role in mediating the responsiveness of the gene to AbCon.


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Fig. 5.   Effect of beta e3 mutation on the 215-bp beta -MHC promoter: activity in NC hearts (A) and responsiveness to AbCon (B). The beta e3 mutation on the 215-bp fragment was associated with a significant decrease in beta -MHC promoter activity relative to the wild-type activity in the NC heart (A). However, its responsiveness to AbCon was not reduced (B). Thus the beta e3 element is a positive regulatory element in the normal expression of the beta -MHC gene but does not play a role in responsiveness of the gene to AbCon. Values are means ± SE; n = 10 for each NC group and as reported in Table 5 for the AbCon groups. *P < 0.05 vs. WT.

Again the heart relative mass (ventricle weight-to- body weight ratio) and percent beta -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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Table 5.   Relative increase in ventricular weight-to-body weight ratios and percent beta -MHC mRNA expression in AbCon groups after injection with the specified 215-bp beta -MHC-pGL3 promoter-reporter construct

Nuclear Extract Interaction With Specific Cis-Elements on the beta -MHC Promoter

DNA gel mobility shift assays were performed to examine cardiac nuclear extract interaction with specific cis-elements on the 408-bp beta -MHC promoter, such as beta e1, beta e2, GATA, and beta e3 elements, and to determine whether these interactions were altered under the AbCon condition. In addition, these assays were used to test the effectiveness of mutations designed to disrupt specific nuclear protein binding sites on the basis of direct binding capacity or competition with the wild-type oligonucleotides. Our results on the interactions between beta e2, GATA, or beta e3 positive elements and cardiac nuclear extract (Fig. 6) indicate that specific complexes formed when NC or AbCon heart extracts were used; however, there was no significant difference in binding between the two groups (Fig. 6). Also these results show that the mutations were effective in disrupting the binding sites; in fact, all mutant oligonucleotides competed much less effectively than the wild-type oligonucleotides for binding when used in 100-fold molar excess to the probe. These findings indicate that, after 12 days of hypertension, hearts do not upregulate the expression of transacting factors interacting with the beta e2, GATA, and beta e3 elements. These results confirm our mutation analyses of the 408-bp beta -MHC promoter (Fig. 4), whereby simultaneous mutations of the beta e2/beta e3 and GATA elements significantly reduced its activity in the NC hearts but did not blunt its responsiveness to AbCon.


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Fig. 6.   Cardiac nuclear extract (NE) binding to beta e2, GATA, and beta e3 cis-regulatory elements using DNA gel mobility shift assays. A: beta e2 binding. Lane 1, no binding was detected after NE interaction with 32P-labeled mutant double-stranded beta e2 oligonucleotide. Lanes 2-5, NE interaction with wild-type beta e2; binding resulted in formation of 3 specific complexes (C1, C2, and C3) that were competed off with 100-fold molar excess of cold self (lane 3); competition failed with 100-fold excess of mutant (Mut) double-stranded beta e2 (lane 4) or palindromic thyroid response element (TRE) double-stranded oligonucleotide (lane 5), which is of similar size but unrelated to the beta e2 sequence. Lanes 6 and 7, beta e2 binding with NE from NC and AbCon (AC) hearts, respectively. B: GATA binding. Lanes 1-4, NE interaction with the GATA-like element. Binding resulted in formation of 2 specific complexes (C1 and C2) that were competed off with 100-fold molar excess of cold self (lane 2); competition was not as effective with 100-fold excess of mutant double-stranded GATA (lane 3) or palindromic TRE sequence (lane 4), which is of similar size but unrelated to the GATA sequence. Lanes 5 and 6, GATA binding with NE from NC and AbCon hearts, respectively. C: beta e3 binding. Lanes 1-4, NE interaction with the beta e3 element. Binding resulted in formation of >= 2 specific complexes (C1 and C2) that were effectively competed off with 100-fold molar excess of cold self (lane 2); competition was not as effective with 100-fold excess of mutant double-stranded beta e3 (lane 3) or palindromic TRE sequence (lane 4), which is of similar size but unrelated to the beta e3 sequence. Lanes 5 and 6, beta e3 binding with NE from NC and AbCon hearts, respectively. In gels, density of the shifted bands was determined by scanning densitometry (ImageQuant, Molecular Dynamics). Bar graph represents data (means ± SE) from 6 independent observations per group, whereby each n consisted of a pool from heart tissue of >= 5 rats. For all competition assays, a mix of NC and AbCon cardiac NE was used for bindings. SU, arbitrary scanning unit; Comp, competitor cold double-stranded oligonucleotides used at 100-fold molar excess to the 32P-labeled probe. See Table 2 for oligonucleotide sequences.

In addition to beta e2, GATA, and beta e3, we also examined the binding to the beta 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 beta e1 sense strand increased by ~26% under AbCon conditions (P < 0.05). beta 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 beta e1 element was mutated in the -408-bp beta -MHC promoter construct (Fig. 4). When this element was deleted, as in the -299-bp beta -MHC promoter (Fig. 3), or mutated, as in the -408-bp beta -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 beta e1 in the AbCon induction of the promoter activity. If beta e1 were a player in the AbCon-induced beta -MHC promoter upregulation, one might expect to find a decreased repressor activity/nuclear extract-beta e1 interaction in AbCon hearts, but that was not the case. In contrast, a trend for the opposite was observed, i.e., increased beta e1 binding (Fig. 7). The significance of this observation remains unclear; thus more research is needed in this area.


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Fig. 7.   Cardiac NE binding to beta e1 and beta -MHC TATA cis-regulatory elements using DNA gel mobility shift assays. A: beta e1 sense strand (ss) binding. Lanes 1-5, NE interaction with the single-stranded beta e1 ss. Binding resulted in formation of >= 2 specific complexes (C1 and C2) that were effectively competed off with 100-fold molar excess of cold self (lane 2); competition was not as effective with use of 100-fold excess of mutant beta e1 ss (lane 3), double-stranded (ds) beta e1 wild type (lane 4), or double-stranded palindromic TRE (lane 5). Lanes 5 and 6, beta e1 ss binding to NE from NC and AbCon hearts, respectively. B: beta -MHC TATA binding. Lanes 1-3, NE interaction with the beta -MHC TATA box. Binding resulted in formation of 1 shifted band (C1) that was effectively competed off with 100-fold molar excess of cold self (lane 2); competition was not as effective with use of 100-fold excess of double-stranded beta e2 element (lane 3), a TATA-unrelated sequence. Lanes 4 and 5, beta -MHC TATA binding to NE from NC and AbCon hearts, respectively. In gels, density of the shifted bands was determined by scanning densitometry (ImageQuant, Molecular Dynamics). Bar graph represents data (means ± SE) from 6 separate n per group, where n consisted of a pool from heart tissue of >= 5 rats. For all competition assays, a mix of NC and AbCon cardiac NE was used for the binding. See Table 2 for oligonucleotide sequences.

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 beta -MHC promoter, a gel mobility shift assay was used to test the interaction between the beta -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 beta -MHC promoter response to AbCon (see below).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -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 beta -MHC promoter deletion constructs containing the rat or human beta e2 element under in vitro and in vivo conditions. When promoter/reporter constructs were transfected into cultured fetal rat cardiomyocytes, the human beta -reporter was expressed more than threefold above the equivalent rat construct. However, when these same beta -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 beta -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 beta -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 alpha -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 beta -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) beta -MHC promoter (Fig. 1). These results make our model suitable to study transcriptional regulation of the beta -MHC promoter in the hypertensive heart. In contrast, the alpha -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 alpha -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 beta -MHC promoter activity is expressed as the beta -to-alpha ratio of reporter gene expression in NC and hypertensive rodent hearts.

Deletion analysis of the beta -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 beta -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 beta -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): beta e2, beta e3, and C-rich are positive regulators of beta -MHC gene expression that have been recognized in cardiac and skeletal muscle cell culture studies (41). In addition, the beta e1 element and at least one thyroid response element (TRE) have been identified as negative regulators of beta -MHC gene expression (7, 8, 44). The results of this study confirm other findings that beta e2 and beta e3 are positive regulatory elements (19), whereas beta e1 is a repressor element (7) regulating the expression of the beta -MHC gene in the normal intact heart.

Of particular interest in the context of hypertension regulation are the beta e2 and beta e3 elements, since they are positive regulators of beta -MHC gene expression. The beta 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 beta 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 beta -MHC gene activation by alpha -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 beta e2 element in the activation of beta -MHC gene transcription in cardiomyocytes in vitro (41).

Recently, a GATA element that is located directly adjacent to the beta e2 element has been implicated as an important regulator of beta -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: beta e2 (-285 to -269 bp), part of beta 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 beta e2 and beta e3 elements did not change aortic constriction-stimulated transcription; however, mutation of the GATA element, which is adjacent to the beta 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 beta -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 beta e2/beta e3 -408-bp double mutant nor the beta e2/beta 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 alpha -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 beta -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 beta -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 beta -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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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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