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(Circulation. 1997;96:3943-3953.)
© 1997 American Heart Association, Inc.

Articles

cis-Acting Sequences That Mediate Induction of ß-Myosin Heavy Chain Gene Expression During Left Ventricular Hypertrophy due to Aortic Constriction

Koji Hasegawa, MD, PhD; Soo Jin Lee, BS; Shawn M. Jobe, BS; Bruce E. Markham, PhD; ; Richard N. Kitsis, MD

From the Cardiovascular Division, Departments of Medicine and Cell Biology, Albert Einstein College of Medicine, Bronx, NY (K.H., S.J.L., R.N.K.), and the Department of Cell Biology, Parke-Davis Pharmaceutical Research Division, Warner Lambert Co, 2800 Plymouth Rd, Ann Arbor, Mich (S.M.J., B.E.M.).

Correspondence to Richard N. Kitsis, Departments of Medicine (Cardiology) and Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY 10461. E-mail kitsis{at}aecom.yu.edu

	Abstract

Top Abstract Introduction Materials and Methods Animal Surgery and Tissue... RNA Analysis Statistical Analyses Results Transactivation of a ~3542... ß-MHC GATA Site Can... Discussion References

 
Background Marked alterations in the expression of specific genes occur during the development of cardiac hypertrophy in vivo. Little is known, however, about the cis-acting elements that mediate these changes in response to clinically relevant hypertrophic stimuli, such as hemodynamic overload, in intact adult animals.

Methods and Results The left ventricular expression of a directly injected reporter gene driven by 3542 bp of rat ß-myosin heavy chain (ß-MHC) promoter was increased 3.0-fold by aortic constriction (P<.005), an increment similar to the 3.2-fold increase in the level of the endogenous ß-MHC mRNA in the same left ventricles. Subsequent analysis identified a 107-bp ß-MHC promoter sequence (-303/-197) sufficient to convert a heterologous neutral promoter to one that is activated by aortic constriction. These sequences contain two M-CAT elements, which have previously been demonstrated to mediate inducible expression during ₁-adrenergic–stimulated hypertrophy in cultured neonatal cardiac myocytes, and a GATA element. Although simultaneous mutation of both M-CAT elements markedly decreased the basal transcriptional activity of an injected 333-bp ß-MHC promoter, it had no effect on aortic constriction-stimulated transcription (3.5-fold increase, P<.005 for both wild type and mutant). In contrast, mutation of the GATA motif markedly attenuated aortic constriction-stimulated transcription (1.6-fold, P=NS) without affecting the basal transcriptional activity. This GATA site can interact with in vitro translated GATA-4 and compete with an established GATA site for GATA-4 binding activity in nuclear extracts from aortic constricted hearts.

Conclusions Basal and aortic constriction-stimulated transcription of the ß-MHC gene is mediated, at least in part, through different mechanisms. A GATA element within ß-MHC sequences -303/-197 plays a role in the transcriptional activation of this gene by aortic constriction.

Key Words: genes • hypertrophy • signal transduction

	Introduction

Top Abstract Introduction Materials and Methods Animal Surgery and Tissue... RNA Analysis Statistical Analyses Results Transactivation of a ~3542... ß-MHC GATA Site Can... Discussion References

 
Hemodynamic overload is a complex physiological stimulus, consisting of humoral, mechanical, and neural components, which elicits marked changes in myocardial structure and function. These include myocyte hypertrophy, chamber dilation, and mechanical failure. Although hemodynamic overload may arise from a wide variety of primary abnormalities, including hypertension, myocardial infarction, and valvular heart disease, its effects on the myocardium play a role in virtually all forms of heart failure. Hemodynamic overload-induced hypertrophy results in marked alterations in cardiac gene expression. These are characterized by transient induction of selected immediate early genes and more prolonged reactivation of cardiac genes whose expression in the ventricle is limited primarily to fetal life. The latter are exemplified by skeletal -actin, ß-myosin heavy chain (MHC), and atrial natriuretic factor (ANF).^1–6 In contrast, expression of -MHC, the major adult isoform of myosin heavy chain in the rodent, is down-regulated^7,8 during hemodynamic overload hypertrophy, demonstrating that these alterations do not represent generalized increases in transcription associated with cardiac growth. Currently, little is known about the precise molecular mechanisms by which hemodynamic overload transduces these changes in gene expression during the development of cardiac hypertrophy.

Previous work to delineate the regulation of cardiac gene expression during myocyte hypertrophy has been performed using transient transfections into primary cultures of neonatal rat cardiac myocytes.^9–16 Treatment of these cells with ₁-adrenergic agonists,^{9,11,12,14,16} various growth factors,^10,13 and mechanical stretch¹⁵ stimulates increases in myocyte volume and reproduces many of the changes in cardiac gene expression characteristic of the hypertrophic program in vivo. Despite these similarities, several features of these cell culture systems raise questions about their ability to model adult cardiac myocytes undergoing hemodynamic overload-induced hypertrophy in vivo. First, myocardial development is not complete at birth and differences in gene expression exist between neonatal and adult cardiac myocytes (for reviews see refs. 1 to 3). Second, differences in gene expression have been observed between dissociated myocytes in culture and those in the intact heart.¹⁷ Third, the time course for the hypertrophic response in vitro is much more rapid than that observed in vivo. Fourth, it is unclear whether a complex physiological stimulus such as hemodynamic overload can be modeled accurately by a single humoral or mechanical stimulus. For example, although myocardial activation of ₁-adrenergic receptors can lead to cardiac hypertrophy in vivo,¹⁸ there is little evidence that this mechanism plays a critical role in disease-related hypertrophic growth. For all of these reasons, it would be advantageous to have a system in which to study cardiac gene regulation in the adult heart in response to a well recognized and clinically important hypertrophic stimulus such as hemodynamic overload.

To accomplish this, we and others have developed a means of transferring genes into striated muscle by the simple direct injection of naked plasmid DNA in vivo.^19–26 Transfection by this approach occurs specifically in myocytes.^19–22,24 In addition, the expression of an injected construct driven by promoter sequences from a cardiac gene is regulated in parallel with that of the endogenous cardiac gene from which the promoter sequences were derived.^23,25 This approach has been used to investigate the transient induction of c-fos expression in response to intraventricular balloon inflation in isolated perfused hearts.²⁶ In the current study, we investigated the roles of ß-MHC and ANF promoter sequences in mediating induction in the expression of these genes in the left ventricles of intact adult rats during hypertrophy evoked by aortic constriction. These genes were chosen for analysis because the accumulation of their products closely parallels the development of hemodynamic overload-stimulated cardiac hypertrophy.^4,8

	Materials and Methods

Top Abstract Introduction Materials and Methods Animal Surgery and Tissue... RNA Analysis Statistical Analyses Results Transactivation of a ~3542... ß-MHC GATA Site Can... Discussion References

 

Plasmids
The reporter plasmid p-3542ß-MHCluc was constructed by cloning a BamHI-XmnI fragment encompassing nucleotides -3542 to +89 relative to the transcriptional start site of the rat ß-MHC gene (generous gift of Dr. John G. Edwards, New England Regional Primate Center) into BamHI-SmaI digested pXP2,²⁷ containing the firefly luciferase (luc) coding region and SV 40 splicing and polyadenylation signals. Reporter plasmids carrying further 5' deletions of this promoter (with the same 3' end at +89) were generated by using HindIII (p-1145ß-MHCluc) or NheI (p-408ß-MHCluc) sites, or by amplifying a part of ß-MHC promoter sequences with polymerase chain reaction (PCR) (p-303ß-MHCluc and p-203ß-MHCluc) and subcloning into a BamHI-BglII-cleaved pXP2. M-CAT and GATA mutations were studied in the context of ß-MHC sequences -333 to +34. p-333wtß-MHCluc (wild type) was generated by PCR. Combined site-directed mutagenesis of the distal (-274 to -280) and proximal (-204 to -210) M-CAT elements was performed in two steps. First, constructs with a mutation in either distal or proximal M-CAT element were generated by PCR. Second, a promoter with mutations in both M-CAT elements (p-333mutA+Bß-MHCluc) was generated by inserting the PflMI fragment of the plasmid containing the proximal M-CAT mutation into the corresponding PflMI sites in the distal M-CAT mutant. p-333mutGATAß-MHCluc was generated by PCR. ptkCAT contains the chloramphenicol acetyltransferase (CAT) gene under the transcriptional control of the herpes simplex virus thymidine kinase promoter sequences -109 to +56.²⁸ p-303/-197tkCAT construct was constructed by amplifying the ß-MHC sequences from nucleotide -303 to -197 with PCR and subcloning this product into XbaI-digested tkCAT. p-3412ANFluc was constructed by fusing the BglII fragment of ANF-pKC7 (generous gift of Dr. Christine E. Seidman, Harvard),²⁹ which includes nucleotides -3412 to +63 of the rat ANF gene, into BamHI-BglII-digested pXP2. pRSVCAT and pRSVluc, containing Rous sarcoma virus (RSV) long-terminal repeat sequences spliced to CAT and luc respectively, have been described previously.^21–23,30 All constructs cloned using PCR were verified by sequencing in both directions. Plasmids were purified by anion exchange chromatography (QIAGEN, Chatsworth, CA), quantitated by measurement of OD₂₆₀, and examined on agarose gels stained with ethidium bromide prior to use.

	Animal Surgery and Tissue Preparation

Top Abstract Introduction Materials and Methods Animal Surgery and Tissue... RNA Analysis Statistical Analyses Results Transactivation of a ~3542... ß-MHC GATA Site Can... Discussion References

 
A total of more than 400 adult female Wistar rats weighing between 175 and 200g were used for these experiments. Cardiac injection was performed as described previously.^21–24 Eighty µl of a solution containing closed, circular plasmid DNA and 3% Evans blue dye (to confirm the intramuscular location of the injection) in 0.9% NaCl were injected through a 27-gauge needle. Plasmid DNA consisted of 5 µg of the reporter construct driven by the cellular promoter of interest and 0.5 µg of pRSVCAT or pRSVluc to correct for variation in transfection efficiency. These dosages were chosen based on preliminary experiments showing that the amount of luciferase activity in cardiac homogenates increases linearly following injection of 0 to 5 µg of this plasmid.²⁴ On the day following injection, animals were subjected to either abdominal aortic constriction or sham operation. Constriction of the aorta above the suprarenal arteries was accomplished by tying a 3-0 silk suture securely around both the aorta and a short segment of a 23 gauge needle, and then removing this needle. In sham operated rats, abdominal aortae were exposed but no ligature placed. Surgical mortality was less than 5% in both sham operated and aortic constricted groups. Post-operative mortality was approximately 40% in the aortic constricted group and 1% in the sham operated group. Animals were sacrificed 12 days following aortic constriction (13 days after injection). This time point was chosen because: a) expression of directly injected genes are maximal and constant 5 to 14 days post-injection;^21–24 b) induction of the endogenous ß-MHC gene in adult rodent ventricles is temporally associated with the development of cardiac hypertrophy and more marked in later stages of hemodynamic overload;^1–4,8 and c) induction of ß-MHC expression is more uniform throughout the ventricle at later time points following aortic constriction compared with its earlier localization in the subendocardium and surrounding intracardiac vessels.³¹ Following sacrifice, hearts were removed, atria and great vessels trimmed, ventricles washed in iced-cold phosphate buffered saline, and left ventricle with interventricular septum, right ventricle, and lungs weighed. Then, the basal one-third of the left ventricle was discarded to avoid contamination with atrial tissue. The middle one-third of the left ventricle was immediately frozen in liquid nitrogen for subsequent RNA analysis, and the apical one-third (~300 mg) was homogenized in 1.5 volumes of ice-cold homogenization buffer³² with a Tissumizer (Tekmar Co., Cincinnati, Ohio). Homogenates were centrifuged at 7000g for 30 minutes at 4°C, and supernatants were used for luc and CAT analyses. In all experiments, aortic constriction was considered successful only if the resulting left ventricular/body weight ratio was 30% higher than that of the mean of the animals subjected to sham operation at the same time. By this criterion, < 10% of animals were excluded from analysis. The protocol was approved by the Ethics Committee of Animal Institute of the Albert Einstein College of Medicine.

Reporter Gene Assays
Both luc and CAT activities were assayed in the same supernatant of cardiac homogenates as described previously.^21–24 Using a Monolight luminometer (Model 2010, Analytical Luminescence Laboratory, San Diego, Calif.), luc activity was measured in 15 µL aliquots of the supernatant. For CAT activity, 15 µL of the supernatant were assayed with an incubation time of 2 hours for samples in which 0.5 µg pRSVCAT was injected. For those in which 5 µg of ptkCAT was injected, 100 µL of the supernatant was assayed with an incubation time of 12 hours. Samples in which CAT conversion was less than 0.5% (background averaged 0.1%) were not reported. By this criterion, no more than two animals in any group were excluded, and no groups were selectively affected. CAT conversions were within the linear range for all determinations reported in this paper. Results are expressed as luc-background (in raw luminometer units)/CAT-background (as percent conversion) in p-ß-MHCluc/p-RSVCAT and p-ANFluc/p-RSVCAT injections and as CAT-background/luc-background in p-tkCAT/p-RSVluc injections.

	RNA Analysis

Top Abstract Introduction Materials and Methods Animal Surgery and Tissue... RNA Analysis Statistical Analyses Results Transactivation of a ~3542... ß-MHC GATA Site Can... Discussion References

 
Northern blot analysis of 10 µg total RNA was performed as previously described.²¹ An isoform specific antisense deoxyoligonucleotide complementary to nucleotides 5846 to 5869 of the rat 3' untranslated region³³ was used to detect ß-MHC mRNA as described previously.²¹ To detect ANF mRNA, a cDNA probe consisting of the PstI fragment of pANF-1,³⁴ which encompasses rat ANF coding region (nucleotides 1 to 580), was radiolabelled by random priming and hybridized in 5x SSC (20x SSC=3 M NaCl, 0.3 M sodium citrate), 1x Denhardt's solution (Ficoll 200 µg/ml, polyvinylpyrrolidone 200 µg/ml, and bovine serum albumin 200 µg/ml), 50 mmol/L sodium phosphate (pH 7.2), 0.2% SDS, and 200 µg/ml denatured salmon sperm DNA at 65°C overnight and then washed in 2x SSC, 0.2% SDS at 65°C for 15 minutes. To normalize for loading and transfer, blots were hybridized with a 5'end-labeled deoxyoligonucleotide probe specific for 28S rRNA as described.³⁵ Abundance of mRNAs were quantified by Phosphorimager analysis (Molecular Dynamics, Sunnyvale, CA). Values of ß-MHC and ANF mRNA were normalized to 28S rRNA.

Electrophoretic Mobility Shift Assays
Double-stranded oligonucleotides were designed that contained GATA motifs from the ß-MHC or B-type natriuretic peptide (BNP) promoters. The sequences of the sense strand of these oligonucleotides were as follows: ß-MHC GATA: 5'-AATGTAAGGGATATTTTTGCTTCACTTTGAG-3';³⁶ ß-MHC GATAmut: 5'-AATGTAAGGtcaATTTTTGCTTCACTTTGAG-3' (mutations in small letters); BNP GATA: 5'-TGTGTCTGATAAATCAGAGATAACCCA-3';³⁷ nonspecific: AGAGCATTTTTGTTGGAGT-3'. Oligonucleotides were synthesized by Life Technology Inc. Nuclear Extracts were prepared from adult hearts of sham or aortic constricted rats and gel shift assays were performed as described earlier.³⁸ Probes were used at 40 fmoles/reaction. In vitro translated GATA-4 was prepared as previously described.³⁸ In competition experiments, all competitors were added at an 800-fold molar excess. Anti-GATA-4 antiserum was kindly provided by Dr. David B. Wilson (Washington University, St. Louis).³⁹

	Statistical Analyses

Top Abstract Introduction Materials and Methods Animal Surgery and Tissue... RNA Analysis Statistical Analyses Results Transactivation of a ~3542... ß-MHC GATA Site Can... Discussion References

 
All data are expressed as means±SEM. The significance of differences between mean values was evaluated by the two tailed Student's t-test and differences considered significant at the P<.05 level.

	Results

Top Abstract Introduction Materials and Methods Animal Surgery and Tissue... RNA Analysis Statistical Analyses Results Transactivation of a ~3542... ß-MHC GATA Site Can... Discussion References

 

	Transactivation of a ~3542 bp ß-MHC Promoter by Aortic Constriction

Top Abstract Introduction Materials and Methods Animal Surgery and Tissue... RNA Analysis Statistical Analyses Results Transactivation of a ~3542... ß-MHC GATA Site Can... Discussion References

 
To determine whether hemodynamic overload stimulates the transcriptional activities of the upstream regulatory sequences of ß-MHC and ANF genes, we evaluated the expression of luciferase reporter genes driven by 3542 bp of rat ß-MHC or 3412 bp of rat ANF 5' flanking sequences in control and hemodynamically overloaded ventricles. Gene transfer was accomplished by directly injecting plasmids p-3542ß-MHCluc or p-3412ANFluc into the left ventricular myocardium of the intact rats. In each case, a small quantity of a second plasmid, pRSVCAT was co-injected as an internal control for transfection efficiency and generalized changes in gene expression between the basal and induced states. On the day following gene transfer, animals were subjected to sham operation or to aortic constriction. Twelve days later, luciferase and CAT activities and steady state levels of the endogenous ß-MHC and ANF mRNAs were assessed in left ventricular homogenates.

As shown in Fig 1A, the mean ratio of left ventricular weight to body weight (LV/BW) was 71% higher in aortic constricted as compared with sham operated animals (P<.001). Among 14 aortic constricted animals, only one was excluded from analysis due to insufficient left ventricular hypertrophy, defined arbitrarily as an increase in LV/BW < 30%. The mean levels of endogenous ß-MHC and ANF mRNAs (normalized to those of 28S rRNA) were 3.2-fold (P<.001) and 10-fold (P<.001) higher respectively in the left ventricles of aortic constricted as compared with sham operated animals (Fig 1B). Thus, this model of aortic constriction resulted in marked left ventricular hypertrophy and induction in the expression of ß-MHC and ANF genes.

View larger version (24K): [in this window] [in a new window]   Figure 1. Transactivation of a 3542 bp rat ß-MHC promoter by aortic constriction in adult rat left ventricular myocardium. A. Left ventricular/body weight ratios of individual sham operated (open circles) and aortic constricted (closed circle) rats. B. Representative Northern blot showing steady state levels of ß-MHC mRNA, ANF mRNA, and 28S rRNA in individual left ventricles of sham operated (lanes 1 to 5) and aortic constricted (lanes 6 to 10) rats. C. Relative ratios of luc/CAT activities following co-injection of 5 µg of luciferase reporters driven by the indicated cellular promoter and 0.5 µg pRSVCAT into left ventricles of rats which were subsequently subjected to sham operation (open bars) or aortic constriction (closed bars) for 12 days. Data are presented as mean±SEM with the mean value of the sham group for each construct set at 1.0. The mean absolute luciferase activities in sham operated animals injected with p-3542ß-MHCluc and p-3412ANFluc were 1.6% and 1.2% respectively of that resulting from injection with pRSVluc. The actual CAT activity of co-injected pRSVCAT did not differ significantly between the sham-operated and aortic-constricted groups in any experiment.

In animals injected with p-3542ß-MHCluc, the mean ratio of luciferase activity to CAT activity (luc/CAT) was 3.0-fold higher in the left ventricles of animals subjected to aortic constriction as compared with sham operation (P<.005; Fig 1C). This effect cannot be attributed to differential effects of hemodynamic overload on the posttranscriptional handling of luc or CAT mRNA or protein because luc/CAT was similar in the left ventricles of aortic constricted and sham operated animals co-injected with pRSVluc and pRSVCAT (Fig 1C). Therefore, increases in the expression of p-3542ß-MHCluc resulting from aortic constriction are due to augmentation of the transcriptional activity of these ß-MHC promoter sequences. This effect is sequence-specific as illustrated by the absence of hemodynamic overload-stimulated increases in transcription directed by the herpes simplex virus thymidine kinase promoter (Fig 3), and a ß-MHC promoter deletion mutant (p-203ß-MHCluc, Fig 2B). Thus, 3542 bp of rat ß-MHC upstream sequence contain sufficient information to increase transcription in response to aortic constriction to a magnitude similar to that exhibited by steady state levels of endogenous ß-MHC mRNA.

View larger version (10K): [in this window] [in a new window]   Figure 3. Conferral of hemodynamic overload-responsiveness onto a heterologous neutral promoter by rat ß-MHC promoter sequences -303/-197. Relative ratios of CAT/luc activities following co-injection of 5 µg of ptk-CAT or p-303/-197tkCAT and 0.5 µg pRSVluc into left ventricles of rats which were subsequently subjected to sham operation (open bars) or aortic constriction (closed bars) for 12 days. Data are presented as mean±SEM with the mean value of the sham group for each construct set at 1.0.

View larger version (31K): [in this window] [in a new window]   Figure 2. Deletion analysis of the transcriptional activities of rat ß-MHC promoter sequences in rat left ventricles during hemodynamic overload-induced hypertrophy. A. Basal transcriptional activity as indicated by the relative ratios of luc/CAT activities 5 days following co-injection of 5 µg of each luciferase construct and 0.5 µg pRSVCAT into left ventricles of rats. Data are presented as mean±SEM with the activity of the 3542 bp ß-MHC promoter set at 100%. B. Inducible activity as indicated by the relative ratios of luc/CAT activities following co-injection of 5 µg of each luciferase construct and 0.5 µg pRSVCAT into left ventricles of rats subsequently subjected to sham operation (open bars) or aortic constriction (closed bars) for 12 days. Data are presented as mean±SEM with the mean value of the sham group for each construct set at 1.0.

We also evaluated the hemodynamic overload-responsiveness of a promoter consisting of 3412 bp of rat ANF 5' flanking region. Although hemodynamic overload results in marked ventricular expression of the endogenous ANF gene, experiments in transgenic mice harboring reporter constructs driven by various rat ANF sequences have demonstrated the absence of inducible reporter expression in left ventricles following aortic constriction.⁴⁰ Injection of p-3412ANFluc resulted in similar luc/CAT in the left ventricles of sham operated and aortic constricted rats (Fig 1C) despite the 10-fold increase in steady state levels of endogenous ANF mRNA in these ventricles (Fig 1B). The fidelity of the construct itself was confirmed by demonstrating a 5.2±0.3-fold induction in reporter expression in response to 100 µM of phenylephrine following transfection into cultured neonatal rat cardiac myocytes (data not shown). Therefore, the results of the gene injection experiments are in agreement with those obtained in transgenic mice and demonstrate the inability of these 3412bp of rat ANF promoter to mediate hemodynamic overload responsive expression of ANF gene in the rat ventricular myocardium.

Analysis of the ß-MHC Promoter
To identify which DNA sequences mediate hemodynamic overload stimulated increases in ß-MHC transcription, nested 5' deletions were created in the rat 3542 bp ß-MHC promoter. Promoters with 5' termini ranging from -3542 to -203 upstream of the ß-MHC cap site resulted in easily detectable amounts of luciferase in adult cardiac myocytes in vivo (Fig 2A). The ability of each construct to respond to hemodynamic overload was evaluated in a series of experiments in which its transcriptional activities in sham operated and aortic constricted hearts were compared side by side according to the protocol described above (Fig 2B). For each construct tested, the mean LV/BW was 60% higher in aortic constricted as compared with sham operated animals indicating the adequacy of the hemodynamic overload stimulus. Deletions to -303 resulted in no significant decrease in inducibility, demonstrating that sequences within the p-303ß-MHCluc construct are sufficient to drive inducible expression. In addition, p-303ß-MHCluc showed the highest level of inducibility among the five constructs tested, suggesting that important elements which mediate inducible expression lie 3' to -303. In contrast, deletion from -303 to -203 resulted in complete loss of inducible expression. This result cannot be attributed to a loss of basal transcriptional activity as p-203ß-MHCluc expressed levels of luciferase in the basal state which exceeded those of another ß-MHC construct, p-1145ß-MHCluc, which remained inducible. Thus, sequences -303/-203 are necessary for hemodynamic overload-stimulated increases in the transcription of p-303ß-MHCluc.

To determine the ability of sequences -303/-197 to confer aortic constriction-inducible expression onto a heterologous neutral promoter in the absence of other ß-MHC sequences, a construct containing a single copy of these sequences fused in the forward orientation to sequences -109 to +56 from the herpes simplex virus thymidine kinase gene (p-303/-197tkCAT) was assessed (Fig 3). While the transcriptional activity of the parental plasmid p-tkCAT was similar in control and hemodynamically overloaded left ventricles, that of p-303/-197tkCAT was 70% higher in the left ventricles of aortic constricted as compared with sham operated animals (P<.005). Thus, rat ß-MHC sequences -303/-197 are sufficient to convert a neutral promoter to one which is hemodynamic overload-responsive.

Role of M-CAT and GATA Elements
Sequences -303/-197 of the rat ß-MHC promoter contain M-CAT and GATA motifs (Fig 4A). M-CAT elements play a role in cardiac and skeletal muscle-specific expression in many striated muscle genes including cardiac troponin T,⁴¹ skeletal -actin,^12,13 -MHC,⁴² as well as ß-MHC^11,43–45 (reviewed in ref. 46). Moreover, the proximal M-CAT element in the rat ß-MHC gene has been shown to be both necessary and sufficient for transactivation of this gene during hypertrophy induced by ₁-adrenergic agonists in cultured neonatal cardiac myocytes.¹¹ To test the role of M-CAT elements in hemodynamic overload-induced activation of the ß-MHC promoter in the setting of adult cardiac myocytes in vivo, mutations previously demonstrated to abolish the binding of nuclear proteins^11,47 were introduced in the context of rat ß-MHC sequences -333 to +34 (p-333mutA+Bß-MHCluc). Both M-CAT elements were mutated simultaneously to eliminate the possibility of redundancy. The expression of this construct was compared with that of the corresponding wild type construct (p-333wtß-MHCluc) in the left ventricles of sham operated and aortic constricted rats. As shown in Fig 4B, mutation of M-CAT elements decreased transcriptional activity in sham operated ventricles by 82% (P<.005). In contrast, expression of both p-333wtß-MHCluc and p-333mutA+Bß-MHCluc were induced 3.5-fold by aortic constriction (P<.005 for both; Fig 4C). Thus, in the context of the 333 bp ß-MHC promoter, M-CAT elements are required for full levels of transcription in the basal state but not for transcription induced by hemodynamic overload.

View larger version (21K): [in this window] [in a new window]   Figure 4. Role of M-CAT and GATA elements in basal and hemodynamic overload-inducible ß-MHC expression. A. Sequence of the rat ß-MHC promoter from -303 to -197 with cis-acting regulatory motifs underlined and base changes introduced into p-333mutA+Bß-MHCluc and p-333mutGATAß-MHCluc indicated below the sequence. B. Basal transcriptional activity as indicated by the relative ratios of luc/CAT activities following co-injection of 5 µg of p-333wtß-MHCluc (wt), p-333mutA+Bß-MHCluc (mut A+B), or p-333mutGATAß-MHCluc (mut GATA) and 0.5 µg pRSVCAT into left ventricles of rats subjected to sham operation the following day and sacrificed 12 days later. Data are presented as the mean±SEM with the mean value of p-333wtß-MHCluc set at 100%. C. Inducible activity as indicated by the relative ratios of luc/CAT activities following co-injection of 5 µg of each luciferase construct and 0.5 µg pRSVCAT into left ventricles of rats subsequently subjected to sham operation (open bars) or aortic constriction (closed bars) for 12 days. Data are presented as mean±SEM with the mean value of the sham group for each construct set at 1.0.

We next examined the role of the GATA motif. GATA elements have been shown to be important for cardiac-specific transcription in the context of many other cardiac genes including -MHC,³⁸ cardiac troponin T,³⁹ myosin light chain 1/3⁴⁸ and B-type natriuretic peptide (BNP).³⁷ A mutation in the core GATA sequence that was previously demonstrated to abolish binding of nuclear proteins in other genes was studied in the context of -333 bp ß-MHC promoter (p-333mutGATAß-MHCluc). While this mutation did not affect basal transcription (Fig 4B), it markedly reduced hemodynamic overload-inducible transcription (1.6-fold, NS for p-333mutGATAß-MHCluc as compared with 3.5-fold, P<.005 for p-333wtß-MHCluc; Fig 4C). These findings indicate that the GATA sequences play a role in mediating the induction of ß-MHC expression during hemodynamic overload hypertrophy in vivo.

	ß-MHC GATA Site Can Interact with GATA-4 in Aortic Constricted Hearts

Top Abstract Introduction Materials and Methods Animal Surgery and Tissue... RNA Analysis Statistical Analyses Results Transactivation of a ~3542... ß-MHC GATA Site Can... Discussion References

 
To determine if the GATA motif in the ß-MHC promoter can interact specifically with GATA-4, electrophoretic mobility shift assays (EMSA) were performed. In vitro translated GATA-4 was probed with a radiolabeled oligonucleotide containing the ß-MHC GATA site in the presence or absence of competitor DNAs (Fig 5A, lanes 1 to 4). Among three retarded bands, competition EMSAs revealed that only the slowest migrating band represents GATA sequence-specific binding as evidenced by the fact that it was competed by an excess of unlabeled ß-MHC GATA oligonucleotide (lane 2) or by an oligonucleotide containing a previously demonstrated GATA site in the BNP promoter (lane 3), but not by the same amount of a nonspecific oligonucleotide (lane 4) or by an oligonucleotide containing the ß-MHC GATA site into which point mutations (Fig 4A) that ablate hemodynamic overload responsiveness had been introduced (data not shown). In addition, the retarded band represents an interaction of the probe with GATA-4 because it was absent in unprogrammed lysate (data not shown). To compare the mobility of this band with complexes formed on an established GATA site, in vitro translated GATA-4 was probed with an oligonucleotide encompassing the BNP GATA motif (Fig 5A, lanes 5 to 8). Two GATA sequence specific interactions were observed with the slower one similar in mobility to that formed on the ß-MHC GATA site (compare lanes 1 and 5). Competitions with an excess of unlabeled BNP GATA competed both this slower band as well as a faster migrating complex (lane 6). In contrast, the same amount of ß-MHC GATA oligonucleotide competed only the slower complex (lane 7), while nonspecific oligonucleotide competed neither band (lane 8). Of note, the intensity of the slow mobility complex increased when in vitro translated GATA-4 was incubated for 72 hours at 4°C prior to use compared with freshly prepared protein (data not shown). Thus, these data demonstrate that the ß-MHC GATA site can interact specifically with GATA-4 in vitro and the mobility of the resulting band is similar to that of the slower of the two bands resulting from the specific interaction of GATA-4 with the BNP GATA site. These data also suggest that GATA-4 may bind the ß-MHC GATA site as a multimer.

View larger version (28K): [in this window] [in a new window]   Figure 5. Analysis of interactions between the ß-MHC GATA site and GATA-4. A. EMSA studies in which in vitro translated GATA-4 was probed with a radiolabeled oligonucleotide containing the ß-MHC GATA (ß-GATA; lanes 1 to 4) or BNP GATA (lanes 5 to 8) sites. Unlabelled competitor DNAs were present at an 800 fold molar excess as indicated: ß-MHC GATA (ß-GATA) lanes 2 and 7; BNP GATA lanes 3 and 6; and nonspecific oligonucleotide (see Materials and Methods) lanes 4 and 8. The arrow indicates the complex corresponding to the GATA-specific interaction between the ß-MHC GATA site and GATA-4. B. Nuclear extract from aortic constricted heart was probed with radiolabeled oligonucleotide containing the BNP GATA site in the presence of 4 µL of either preimmune (PI) or GATA-4 (G4) antiserum as indicated. Competitor DNAs are as indicated. The arrow indicates the position of the GATA-4-dependent supershifted band (SS, lanes 2 and 4).

To determine if the ß-MHC GATA site binds GATA proteins in the context of the normal and hemodynamically overloaded adult myocardium, EMSAs were performed with nuclear extracts from sham operated and aortic constricted hearts. Multiple bands were observed following the incubation of sham operated nuclear extract with ß-MHC GATA-4. Competition studies revealed that all of these protein-DNA interactions were nonspecific (data not shown). With aortic constricted extract, multiple nonspecific bands were observed as well as one band representing GATA-specific binding (data not shown). The intensity of the latter was quite faint, however, precluding supershift experiments to identify the component protein(s). This is not surprising given the the results of the studies using in vitro translated GATA-4 (Fig 5A) suggesting that the ß-MHC GATA site has a relatively low affinity for GATA-4. Therefore, to circumvent this problem and to test whether the ß-MHC GATA site could interact with GATA-4 in the context of an aortic constricted nuclear extract, we used the BNP oligonucleotide as a probe (Fig 5B). Several bands were observed when aortic constricted heart extract was probed with the BNP GATA. Addition of preimmune serum to the reaction did not significantly affect the interactions (lane 1). In contrast, the addition of GATA-4 antiserum resulted in a supershift, establishing that GATA-4 is one of the components (lane 2). This supershift was competed by unlabeled ß-MHC GATA oligonucleotide (lane 3) but not by an oligonucleotide identical except for point mutations in the ß-MHC GATA site (Fig 4A) that ablate hemodynamic overload responsiveness (lane 4). These data indicate that the ß-MHC GATA site can bind in a sequence specific manner to GATA-4 in nuclear extract from aortic constricted hearts.

	Discussion

Top Abstract Introduction Materials and Methods Animal Surgery and Tissue... RNA Analysis Statistical Analyses Results Transactivation of a ~3542... ß-MHC GATA Site Can... Discussion References

 
A combination of humoral, mechanical, and neural factors, referred to collectively as hemodynamic overload, plays a key role in the pathogenesis of virtually all disorders of heart muscle. This stimulus elicits hypertrophy of cardiac myocytes, characterized by increases in cell volume and the synthesis of additional sarcomeres. After a highly variable time interval, these events are often followed by the deterioration of contractile function. A long sought after goal has been to understand the molecular basis by which hemodynamic overload elicits these pathological effects. Since the expression patterns of specific cardiac genes change in temporal association with these events,^1–8 it has been postulated that the signaling pathways that control the expression of these genes overlap significantly with those that mediate these structural and functional changes. For this reason, significant efforts have been directed toward understanding the regulation of these genes both in the basal state and in response to various perturbations.^9–16 These studies, however, have been limited largely to cultured cardiac myocytes from neonatal rats and to purely humoral or relatively simple mechanical stimuli that can be replicated in a culture dish. We have established a system that permits one to delineate the mechanisms by which hemodynamic overload, in all its complexity, modulates gene expression during hypertrophy of adult cardiac myocytes in vivo. This system has been employed to study the regulation of the ß-MHC gene, whose accumulation in myocytes of adult rodent ventricles closely parallels the development of cardiac hypertrophy following aortic constriction.^4,8 These experiments demonstrate that induction of ß-MHC expression during hemodynamic overload hypertrophy is regulated at the level of transcription and that a GATA element within ß-MHC sequences -303/-197 plays a role in mediating this response.

Direct evidence for the transcriptional regulation of cardiac genes during hypertrophy has been provided by nuclear run-on assays evaluating the response of the -skeletal actin gene to ₁-adrenergic agonists in cultured neonatal cardiac myocytes.⁴⁹ In addition, transient transfections of reporter constructs into these cells have provided indirect evidence that transcription plays a role in the increases in ß-MHC,¹¹ ventricular myosin light chain 2,^9,14 and ANF^9,10 expression in response to ₁-adrenergic agonists^9,11,14 and endothelin-1.¹⁰ Although nuclear run-on assays have been successfully performed using neonatal cardiac tissue,⁵⁰ the difficulty in isolating transcriptionally active nuclei from adult cardiac tissue has impeded direct evaluation of the effect of hemodynamic overload on the transcription of specific genes in the context of the intact adult heart. The reporter assays in this study provide the first evidence, albeit indirect, that hemodynamic overload increases ß-MHC transcription. Moreover, the quantitatively similar increments in the transcriptional activities of ß-MHC regulatory sequences and in the steady state levels of endogenous ß-MHC mRNA suggest that transcriptional regulation plays the predominant role in the stimulation of ß-MHC expression by hemodynamic overload. Interestingly, the expression of a reporter gene driven by 3412 bp of ANF upstream sequence was not increased by hemodynamic overload despite marked induction in the expression of the endogenous ANF gene. This result, which is consistent with the findings of transgenic experiments,⁴⁰ suggests that additional cis-acting sequences are required for hemodynamic overload-stimulated ANF transcription and/or increases in endogenous ANF mRNA levels occur through posttranscriptional mechanisms.

The transcriptional activities of nested 5' ß-MHC promoter deletions to -303 retained full hemodynamic overload-respnsiveness. In contrast, hemodynamic overload did not induce the expression of a transcriptionally active mutant with 5' terminus at -203. Although these findings by no means exclude the existence of hemodynamic overload-responsive sequences elsewhere in the rat ß-MHC gene, they demonstrate clearly that sequences -303/-203 are necessary for hemodynamic overload-inducibility in the context of the -303 mutant. In addition, although not as efficient as in its normal context, a single copy of the 107-bp fragment, -303/-197, was able to confer hemodynamic overload-responsiveness onto a heterologous neutral promoter. Thus, these sequences are sufficient to transduce signals generated by hemodynamic overload in adult cardiac myocytes. Taken together, these findings suggest that sequences -303/-197 are involved in mediating hemodynamic overload-stimulated transcription of the ß-MHC gene.

The M-CAT motif is present in the promoters of several striated muscle-specific genes where it functions as a positive regulatory element.^41–46 Rat ß-MHC sequences -303/-197 contain two such M-CAT elements: distal (-274 to -280) and proximal (-204 to -210). The distal M-CAT element in the ß-MHC promoter is perfectly conserved among human, rabbit, rat, and mouse and the proximal one, while not conserved, matches the M-CAT consensus sequence exactly.^{11,42–45,51} Both elements have been shown to be binding sites for the transcription factor TEF-1.^42,47,52 Mutation of either element in the context of a truncated ß-MHC promoter diminishes transcription both in cultured cardiac myocytes^11,42–44 and in the hearts of adult transgenic mice.⁴⁵ Our observation that simultaneous disruption of both M-CAT elements in the setting of a 333 bp ß-MHC promoter markedly decreases transcriptional activity is consistent with this data.^41–46 These mutations have no effect on basal transcriptional activity of a ~5000 bp mouse ß-MHC promoter in transgenic mice, however, suggesting that loss of both M-CAT elements can be compensated for by upstream sequences.⁴⁵ Thus, while not indispensable, M-CAT elements appear to play a role in basal transcription of the ß-MHC gene.

Previous work in cell culture models of neonatal cardiac myocyte hypertrophy has delineated potential elements which mediate ₁-adrenergic-, endothelin-1-, or transforming growth factor-ß-inducible expression during hypertrophy.^11–14,16 These include HF-1 in the ventricular myosin light chain promoter,¹⁴ GAG motif in the ANF promoter,¹⁶ and M-CAT elements in the -skeletal actin and ß-MHC promoters.^11–13 The M-CAT element has also been shown to be necessary and sufficient for ₁-adrenergic-stimulated transcription of the ß-MHC gene and necessary for ₁-adrenergic and transforming growth factor-ß-stimulated activation of the -skeletal actin promoter during hypertrophy in cultured neonatal cardiac myocytes.^11–13 In contrast with these in vitro studies, simultaneous mutations in both M-CAT elements, adequate to abrogate binding of nuclear proteins and destroy enhancer function, had no effect on the hemodynamic overload-responsiveness of a 333 bp rat ß-MHC promoter in adult cardiac myocytes in vivo. Thus, even in the setting of a truncated promoter, M-CAT elements appear dispensable for hemodynamic overload-induced transcription. Our data do not rule out the possibility that M-CAT elements still contribute to hemodynamic overload responsiveness, however. In addition, our experiments were limited to a single time point relatively late after the onset of hemodynamic overload; therefore, it remains possible that M-CAT elements contribute to inducible expression at earlier points in time.

Rat ß-MHC sequences -303/-197 also contain a putative GATA element. GATA factors 4, 5, and 6, which are present in cardiac myocytes and/or their progenitors, play important roles in the transcriptional regulation of cardiac genes (reviewed in ref. 53) and heart morphogenesis.^54,55 Although mutation of GATA elements in -MHC,³⁸ cardiac troponin T,³⁹ myosin light chain 1/3⁴⁸ and BNP³⁷ diminishes their transcriptional activities in cardiac myocytes in culture^37,39 and in vivo,^38,48 it had no effect on that of the 333 bp ß-MHC promoter. In contrast, this same GATA mutation markedly attenuated the ability of the ß-MHC promoter to respond to hemodynamic overload. We hasten to point out, however, that although this element clearly mediates the transcriptional response of ß-MHC to hemodynamic overload, it may not be the only element in the -303/-203 ß-MHC fragment that regulates this response. Indeed, given the potential complexities of the hemodynamic overload stimulus and the multitude of pathways that mediate it, it would not be surprising if combinatorial interactions among several elements were required to precisely reconstitute inducible expression during hypertrophy in vivo. In any event, the differential effects of M-CAT or GATA mutations on basal and hemodynamic overload-induced transcription suggest that these processes are mediated, at least in part, through different mechanisms.

The GATA element has been shown to be a binding site of GATA transcription factors in the context of other cardiac genes^37–39,48 and the EMSA studies herein demonstrate that ß-MHC GATA site can also bind to GATA-4 both in vitro and in vivo. Interestingly, the mobility of the complex resulting from the interaction between ß-MHC GATA site and in vitro translated GATA-4 was similar to that of one of the complexes produced by the binding of the BNP GATA site to GATA-4. This complex was less abundant and of slower mobility than the major complex formed between the BNP GATA site and GATA-4. This result suggests that GATA-4 may be binding the ß-MHC GATA site as a higher ordered complex (possibly a dimer) as compared with the majority of its binding to the BNP GATA site (possibly a monomer). Although this interpretation is provisional and needs to be tested directly, it suggests one model by which GATA transcription factors could modulate the basal transcription of certain cardiac genes and the inducible expression of others. In this model, the promoters dependent on GATA for basal transcriptional activity would be activated by the binding of GATA monomers while the higher concentration of GATA factors in the hemodynamically overloaded state³⁸ would drive the formation of multimeric complexes on GATA sites that mediate hemodynamic overload inducible transcription. To understand the precise mechanism by which GATA sequences mediate hemodynamic overload responsive transcription, however, it will be necessary to define exactly which GATA, as well as possibly nonGATA, proteins bind these sequences and the stoichiometry of these interactions in control and hemodynamically overloaded hearts.

The results of our experiments differ in important respects from those obtained in various cell culture models of cardiac myocyte hypertrophy. For example, although a ~3000 bp rat ANF promoter is activated by ₁-adrenergic agonists and endothelin-1 in cultured rat neonatal cardiac myocytes (9,10; and our data not shown), its transcriptional activity is not increased by hemodynamic overload following injection into adult rat hearts (Fig 1C) or in transgenic mice.⁴⁰ Conversely, although rat ß-MHC promoters longer than 303 bp do respond to hemodynamic overload in adult hearts in vivo, even a 673 bp promoter did not respond to stretch-induced hypertrophy in cultured neonatal cardiac myocytes.¹⁵ These discrepancies support the notion that differences exist between the pathways that mediate induction of ß-MHC and ANF expression during hypertrophy due to chronic hemodynamic overload in intact adult animals and short-term stimuli in cultured neonatal cardiac myocytes. These discrepancies may be attributable to differences in the developmental stage of cells (adult versus neonatal), the extracellular environment (in vivo vs. in vitro), or the nature of the hypertrophic stimulus itself (hemodynamic overload versus a single humoral stimulus or stretch). Because cultured neonatal cardiac myocytes provide a relatively simple means of testing a single well defined stimulus in the absence of other confounding factors, they have provided and will undoubtedly continue to provide an important experimental approach for understanding cardiac hypertrophy. Nevertheless, the differences noted above underscore the importance of eventually testing all conclusions in vivo.

Note Added in Proof
While this manuscript was under review, Herzig et al.⁵⁶ reported that GATA-4 is involved in induction of the expression of the angiotensin II type 1a receptor gene in response to hemodynamic overload.

	Selected Abbreviations and Acronyms

 

ANF = artrial natriuetic peptide BNP = B-type natriuetic peptide CAT = chloramphenicol acetyltransferase EMSA = electrophoretic mobility shift assay luc = firefly luciferase LV/BW = ratio or left ventricular weight to body weight MHC = myosin heavy chain PCR = polymerase chain reaction RSV = Rouse sarcoma virus<.>

	Acknowledgments

 
This work was supported by grants from the NIH (HL-02699 to Dr Kitsis; HL-43662 to Dr Markham), and from the American Heart Association, New York City Affiliate (Dr Kitsis). Dr Kitsis is the Charles and Tamara Krasne Faculty Scholar in Cardiovascular Research of the Albert Einstein College of Medicine. We thank Dr Francis Siri for advice regarding the aortic constriction model.

Received July 24, 1997; revision received September 5, 1997; accepted September 25, 1997.

	References

Top Abstract Introduction Materials and Methods Animal Surgery and Tissue... RNA Analysis Statistical Analyses Results Transactivation of a ~3542... ß-MHC GATA Site Can... Discussion References

 

1. Chien KR, Knowlton KU, Zhu H, Chien S. Regulation of cardiac gene expression during myocardial growth and hypertrophy: molecular studies of an adaptive physiological response. FASEB J. 1991;5:3037–3046.[Abstract]
   
2. Nadal-Ginard B, Mahdavi V. Molecular basis of cardiac performance: plasticity of the myocardium generated through protein isoform switches. J. Clin. Invest. 1989;84:1693–1700.
   
3. Schwartz K, Boheler KR, de la Bastie D, Lompre AM, Mercadier JJ. Switches in cardiac muscle gene expression as a result of pressure and volume overload. Am. J. Physiol. 1992;262:R364–R369.[Abstract/Free Full Text]
   
4. Izumo S., Lompre A, Matsuoka R, Koren G, Schwarz K, Nadal-Ginard B, Mahdavi V. Myosin heavy chain messenger RNA and protein isoform transitions during cardiac hypertrophy. J. Clin. Invest. 1987;79:970–977.
   
5. Lee RT., Bloch KD, Pfeffer JM, Pfeffer MA, Neer EJ, Seidman CE. Atrial natriuretic factor gene expression in ventricles of rats with spontaneous biventricular hypertrophy. J. Clin. Invest. 1988;81:431–434.
   
6. Izumo S, Nadal-Ginard B, Mahdavi V. Protooncogene induction and reprogramming of cardiac gene expression produced by pressure overload. Proc. Natl. Acad. Sci. U. S. A.. 1988;85:339–343.[Abstract/Free Full Text]
   
7. Chassagne C., Wisnewsky C, Schwartz K. Antithetical accumulation of myosin heavy chain but not -actin mRNA isoforms during early stages of pressure overload-induced rat cardiac hypertrophy. Circ. Res. 1993;72:857–864.[Abstract/Free Full Text]
   
8. Ojamaa K., Petrie JF, Balkman C, Hong C, Klein I. Posttranscriptional modification of myosin heavy chain gene expression in the hypertrophied rat myocardium. Proc. Natl. Acad. Sci. U. S. A.. 1994;91:3468–3472.[Abstract/Free Full Text]
   
9. Knowlton KU., Baracchini E, Ross S, Harris AN, Henderson SA, Evans SM, Glembotski CC, Chien KR. Co-regulation of the atrial natriuretic factor and cardiac myosin light chain-2 genes during -adrenergic stimulation of neonatal rat ventricular cells-identification of cis sequences within an embryonic and a constitutive contractile protein gene which mediate inducible expression. J. Biol. Chem. 1991;266:7759–7768.[Abstract/Free Full Text]
   
10. Shubeita HE., McDonough PM, Harris AN, Knowlton KU, Glembotski CC, Brown JH, Chien KR. Endothelin induction of inositol phospholipid hydrosis, sarcomere assembly, and cardiac gene expression in ventricular myocytes-a paracrine mechanism for myocardial cell hypertrophy. J. Biol. Chem. 1990;265:20555–20562.[Abstract/Free Full Text]
    
11. Kariya K., Karns LR, Simpson PC. An enhancer core element mediates stimulation of the rat ß-myosin heavy chain promoter by an ₁-adrenergic agonist and activated ß-protein kinase C in hypertrophy of cardiac myocytes. J. Biol. Chem. 1994;269:3775–3782.[Abstract/Free Full Text]
    
12. Karns LR, Kariya K, Simpson PC. M-CAT, CArG, and Sp1 elements are required for ₁-adrenergic induction of the skeletal -actin promoter during cardiac myocyte hypertrophy. Transcriptional enhancer factor-1 and protein kinase C as conserved transducers of the fetal program in cardiac growth. J. Biol. Chem. 1995;270:410–417.[Abstract/Free Full Text]
    
13. MacLellan WR, Lee TC, Schwartz RJ, Schneider MD. Transforming growth factor-ß response elements of the skeletal -actin gene. Combinational action of serum response factor, YY1, and the SV40 enhancer-binding protein, TEF-1. J. Biol. Chem. 1994;269:16754–16760.[Abstract/Free Full Text]
    
14. Zhu H., Garcia AV, Ross RS, Evans SM, Chien KR. A conserved 28-base-pair element (HF-1) in the rat cardiac myosin light-chain-2 gene confers cardiac-specific and -adrenergic-inducible expression in cultured neonatal rat myocardial cells. Mol. Cell. Biol. 1991;11:2273–2281.[Abstract/Free Full Text]
    
15. Sadoshima J., Jahn L, Takahashi T, Kulik TJ, Izumo S. Molecular characterization of the stretch-induced adaptation of cultured cardiac cells. J. Biol. Chem. 1992;267:10551–10560.[Abstract/Free Full Text]
    
16. Ardati A., Nemer M. A nuclear pathway for ₁-adrenergic receptor signaling in cardiac cells. Embo J. 1993;12:5131–5139.[Medline] [Order article via Infotrieve]
    
17. Kitsis RN, Leinwand LA. Discordance between gene regulation in vitro and in vivo. Gene Expresson. 1992;2:313–317.
    
18. Milano CA., Dolber PC, Rockman HA, Bond RA, Venable ME, Allen LF, Lefkowitz RJ. Myocardial expression of a constitutively active 1B-adrenergic receptor in transgenic mice induces cardiac hypertrophy. Proc. Natl. Acad. Sci. U. S. A.. 1994;91:10109–10113.[Abstract/Free Full Text]
    
19. Wolff JA., Malone RW, Williams P, Chong W, Acsadi G, Jani A, Felgner PL. Direct gene transfer into mouse muscle in vivo. Science. 1991;247:1465–1468.
    
20. Lin H, Parmacek MS, Morle G, Bolling S, Leiden JM. Expression of recombinant genes in myocardium in vivo after direct injection of DNA. Circulation. 1990;82:2217–2221.[Abstract/Free Full Text]
    
21. Kitsis RN, Buttrick PM, NcNally EM, Kaplan ML, Leinwand LA. Hormonal modulation of a gene injected into rat heart in vivo. Proc. Natl. Acad. Sci. U. S. A.. 1991;88:4138–4142.[Abstract/Free Full Text]
    
22. Buttrick P. M., Kass A, Kitsis RN, Kaplan ML, Leinwand LA. Behavior of genes directly injected into the rat heart in vivo. Circ. Res. 1991;70:193–198.[Abstract/Free Full Text]
    
23. Buttrick PM, Kaplan ML, Kitsis RN, Leinwand LA. Distinct behavior of cardiac myosin heavy chain gene constructs in vivo-discordance with in vitro results. Circ. Res. 1993;72:1211–1217.[Abstract/Free Full Text]
    
24. Kitsis RN, Buttrick PM, Kass AA, Kaplan ML, Leinwand LA. Gene transfer into adult rat heart in vivo. Methods in Molecular Genetics. 1993;1:374–392.
    
25. Ojamaa K, Klein I. In vivo regulation of recombinant cardiac myosin heavy chain gene expression by thyroid hormone. Endocrinology. 1993;132:1002–1006.[Abstract]
    
26. Aoyagi T, Izumo S. Mapping of the pressure response element of the c-fos gene by direct DNA injection into beating hearts. J. Biol. Chem. 1993;268:27176–27179.[Abstract/Free Full Text]
    
27. Nordeen SK. Luciferase reporter gene vectors for analysis of promoters and enhancers. Biotechniques. 1988;6:454–457.[Medline] [Order article via Infotrieve]
    
28. Edlund T., Walker MD, Barr PJ, Rutter RJ. Cell-specific expression of the rat insulin gene: evidence for role of two distinct 5' flanking elements. Science. 1985;230:912–916.[Abstract/Free Full Text]
    
29. Seidman CE, Wong DW, Jarcho JA, Bloch KD, Seidman JG. Cis acting sequences that modulate atrial natriuretic factor gene expression. Proc. Natl. Acad. Sci. U. S. A.. 1988;85:4104–4108.[Abstract/Free Full Text]
    
30. Gorman CM, Merlino GT, Willingham MC, Pastan I, Howard BH. The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection. Proc. Natl. Acad. Sci. U. S. A.. 1982;79:6777–6781.[Abstract/Free Full Text]
    
31. Schiaffino S., Samuel JL, Sassoon D, Lompre AM, Garner I, Marotte F, Buckingham M, Rappaport L, Schwartz K. Nonsynchronous accumulation of -skeletal actin and ß-myosin heavy chain mRNAs during early stages of pressure overload-induced cardiac hypertrophy demonstrated by in situ hybridization. Circ. Res. 1989;64:937–948.[Abstract/Free Full Text]
    
32. Brasier AR., Tate JE, Habener JF. Optimized use of the firefly luciferase assay as a reporter gene in mammalian cell lines. Biotechnique. 1989;7:1116–1122.[Medline] [Order article via Infotrieve]
    
33. Kraft R, Bravo-Zehnder M, Taylor DA, Leinwand LA. Complete nucleotide sequence of full length cDNA for rat ß cardiac myosin heavy chain. Nucleic Acids Res. 1989;17:7529–7530.[Free Full Text]
    
34. Seidman CE, Duby AD, Choi E, Graham RM, Harber E, Homcy C, Smith JA, Seidman JG. Structure of rat preproatrial natriuretic factor as defined by a complementary DNA clone. Science. 1984;225:324–326.[Abstract/Free Full Text]
    
35. Schreiber-Agus N, Chin L, Chen K, Torres R, Thomson CT, Sacchettini JC, Depinho RA. Evolutionary relationships and functional conservation among vertebrate Max-associated proteins: the zebra fish homolog of Mxi1. Oncogene. 1994;9:3167–3177.[Medline] [Order article via Infotrieve]
    
36. Thompson WR, Nadal-Ginard B, Mahdavi V. A MyoD1-independent muscle-specific enhancer controls the expression of the ß-myosin heavy chain gene in skeletal and cardiac muscle cells. J. Biol. Chem. 1991;266:22678–22688.[Abstract/Free Full Text]
    
37. Grepin C, Dagnino L, Robitaille L, Haberstroh L, Antakly T, Nemer M. A hormone-encoding gene identifies a pathway for cardiac but not skeletal muscle gene transcription. Mol. Cell. Biol. 1994;14:3115–3129.[Abstract/Free Full Text]
    
38. Molkentin JD, Kalvakolanu DV, Markham BE. Transcription factor GATA-4 regulates cardiac muscle-specific expression of the -myosin heavy chain gene. Mol. Cell. Biol. 1994;14:4947–4957.[Abstract/Free Full Text]
    
39. Ip HS, Wilson DB, Heikinheimo M, Tang Z, Ting C, Simon MC, Leiden JM, Parmacek MS. The GATA-4 transcription factor transactivates the cardiac muscle-specific troponin C promoter-enhancer in nonmuscle cells. Mol. Cell. Biol. 1994;14:7517–7526.[Abstract/Free Full Text]
    
40. Knowlton KU, Rockman HA, Itani M, Vovan A, Seidman CE, Chien KR. Divergent pathways mediate the induction of ANF transgenes in neonatal and hypertrophic ventricular myocardium. J. Clin. Invest. 1995;96:1311–1318.
    
41. Farrance IKG, Mar JH, Ordahl CP. Characterization of a promoter element required for transcription in myocardial cells. J. Biol. Chem. 1992;267:17234–17240.