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Transcription factor Sp1 regulates SERCA2 gene expression in pressure-overloaded hearts: a study using in vivo direct gene transfer into living myocardium
Journal of Molecular and Cellular Cardiology 35 (2003) 777–783 www.elsevier.com/locate/yjmcc
Original Article
Transcription factor Sp1 regulates SERCA2 gene expression in pressure-overloaded hearts: a study using in vivo direct gene transfer into living myocardium > Takako Takizawa a, Masashi Arai a,*, Koichi Tomaru a, Norimichi Koitabashi a, Debra L. Baker b, Muthu Periasamy b, Masahiko Kurabayashi a a b
Second Department of Internal Medicine, Gunma University School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma 371-8511, Japan Department of Physiology and Cell Biology, College of Medicine and Health Sciences, Ohio State University, Columbus, OH 43210-1218, USA Received 24 February 2003; accepted 19 March 2003
Abstract Pressure-overload hypertrophy results in downregulation of the sarcoplasmic reticulum Ca2+-ATPase pump encoding SERCA2 gene that regulates Ca2+ uptake and myocardial relaxation. We previously characterized a proximal promoter region containing four Sp1 element consensus sequences (–284 to –72 base pairs (bp)) that was responsible for pressure-overload-induced transcriptional regulation. The purpose of the present study was to determine which of the Sp1 sites was responsible for the downregulation of SERCA2 gene transcription under pressure overload. Using an in vivo direct gene transfer assay, SERCA2 gene transcriptional activity was measured under pressure overload. Site-directed mutagenesis of the four Sp1 sites (I-IV) in the SERCA2 gene promoter (–284 to –72 bp) was performed. Wild-type and Sp1 mutant-luciferase reporter constructs were injected into the left-ventricular apices of pressure overload or sham-operated rats, and Sp1 mRNA and SERCA2 gene-luciferase activity was measured sequentially from 3 to 14 d after surgery. At 5 d, Sp1 mRNA in the pressure-overload rats increased to 124 ± 7% of sham group levels, and pressure-overload-induced SERCA2 transcriptional activity was 15 ± 4% of sham group when all four Sp1 sites remained intact. Mutation of the Sp1 mutant sites I (–196 to –191 bp) and III (–118 to –113 bp) blocked the inhibitory effect of pressure overload and resulted in SERCA2 gene transcriptional activity of 54 ± 15% and 56 ± 7% of sham group, respectively. We conclude that the pressure-overload-induced decrease in SERCA2 mRNA is mediated by Sp1 sites I and III. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: SERCA2; Pressure overload; Cardiac hypertrophy; Sp1; Gene transcription factor; Luciferase assay; In vivo gene transfer
1. Introduction The sarcoplasmic reticulum (SR) plays an important role in the cardiac contraction-relaxation cycle. Myocardial contraction is triggered by Ca2+ release from the SR via ryanodine receptors, and reuptake of Ca2+ via the SR Ca2+-ATPase (SERCA2) pump results in relaxation. Previous reports suggested that pressure overload results in decreases in SERCA2 mRNA, SERCA2 protein concentration, and Ca2+ reuptake function [1–7]. We recently demonstrated that decreases in SERCA2 mRNA levels resulted from downregulation of
> The review of this manuscript was handled by consulting editor, Stephen F. Vatner, M.D. * Corresponding author. Tel.: +81-27-220-8145; fax: +81-27-220-8158. E-mail address: [email protected] (M. Arai).
© 2003 Elsevier Science Ltd. All rights reserved. DOI: 10.1016/S0022-2828(03)00122-6
SERCA2 gene transcription [8] via regulatory sites in the SERCA2 gene promoter (distal site, –1810 to –1110 base pairs (bp); proximal site, –284 to –72 bp). Pressure overload results in altered transcription profiles of various genes. Genes affected by pressure overload include atrial natriuretic peptide (ANP), the angiotensin II type-1a receptor and myocardial lipid metabolism (MCLM) genes, which are under control of transcription factors such as AP-1, CRE, GATA-4, Sp1, and Sp3 [9–12]. While the SERCA2 gene lacks binding sites for AP-1 and Sp3, CRE, and GATA-binding sites are present between –1810 and –1110 bp, and four Sp1 sites are present in the proximal region between –284 and –72 bp. However, the contribution of each individual site in SERCA2 gene transcription regulation during pressure-overload-induced hypertrophy has not been characterized.
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Muscle-specific genes, including cardiac a-actin, skeletal a-actin, b-myosin heavy chain, and SERCA2, require Sp1 for efficient gene expression [13–16]. Further, a1-adrenergic-mediated induction of the skeletal a-actin and ANP genes is dependent on Sp1 and CArG elements [17,18]. As Sp1 appears to play an essential role in the regulation of musclespecific genes, the present study investigated the importance of the proximal regulatory region that contains four putative Sp1-binding sites to SERCA2 gene transcription and attempted to characterize the contribution of each individual Sp1-binding site. By injecting reporter genes into beating hearts, we characterized the role of each individual Sp1binding site on SERCA2 gene transcription in an in vivo model of cardiac hypertrophy.
2. Materials and methods 2.1. DNA probes The following cDNA fragments were used as mRNA hybridization probes: 1. rabbit SR Ca2+-ATPase2 (SERCA2) cDNA [19], nucleotides 1798–3594; 2. rat Sp1 cDNA [20,21], nucleotides 21–925; 3. human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA [22], nucleotides 37–740. 2.2. Reporter gene construction The proximal promoter region of the SERCA2 gene (–284 to –72 bp) contains four putative Sp1-binding sites (Fig. 1A). Mutants were generated using the altered sites II in vitro mutagenesis kit (Promega) to eliminate Sp1-binding sites. Second and third guanine or cytosine nucleotides were replaced with two thymidine nucleotides in each Sp1 consensus sequence (Fig. 1A). Four single mutation (I-IV), double mutation (I + II) and triple mutation (I + II + III) in Sp1 sites were created within the SERCA2 promoter region from –284 to +350 bp relative to the transcription start site and connected to the fireflyluciferase reporter gene. Transcriptional activity of the SERCA2 gene was determined in the various mutant groups and compared with wild-type promoter construct (Fig. 1B). Control vectors containing the CMV promoter (pRL-CMV sea pansy luciferase) were obtained from a commercial source (Promega, Madison, WI, USA) and used for correction of transfection efficiency variation. 2.3. Direct plasmid injection into in vivo myocardium and hemodynamic measurements Plasmids were directly injected into the myocardium of adult Wistar rats as previously described [23,24]. Briefly, 50 µl of normal saline solution containing 10 µg of SERCA2 firefly-luciferase plasmid and 1 µg of pRL-CMV control plasmid were injected via a 30-G needle into the left-
Fig. 1. (A) Nucleotide sequence of the SERCA2 proximal promoter (–284 to –72 bp). The consensus binding sequences for the Sp1 transcription factor (sites I-VI) are indicated. Second and third guanine or cytosine nucleotides were replaced with two thymine nucleotides in each Sp1 consensus sequence by using in vitro site-specific mutagenesis. (B) Plasmid construction. Schematic representation of mutations in individual Sp1 consensus sites (I-VI) and in combinations (I + II and I + II + III). The Sp1 consensus sequences were mutated from consensus GGGCGG to GTTCGG by sitedirected mutagenesis of the –284 to +350 bp SERCA2 promoter. Six constructs of the SERCA2 promoter-luciferase reporter plasmid were used.
ventricular apex. The activities of firefly luciferase and sea pansy luciferase were measured sequentially from a single sample (dual-luciferase reporter assay system, Promega, Madison, WI, USA). The titer of firefly luciferase representing the activity of SERCA2 gene promoter was normalized to sea pansy luciferase activity, an internal standard of transfection efficiency. Abdominal aortic constriction was achieved concomitant to plasmid injection via a silver aortic clip placed between the renal and superior mesenteric arteries [8,25,26]. Sham-operated controls underwent the same processing as experimental animals except for placement of the aortic clip. To determine the time course of transcriptional activity of the SERCA2 gene after pressure overload, rats were sacrificed at various time points (3, 5, 7 and 14 d post-operatively, n = 4 for each time point). Measurements of ascending aortic pressure, leftventricular end-diastolic pressure (LVEDP), dP/dt, and heart rate were performed via the carotid artery using a Millar catheter (3Fr Millar Instruments, Inc., USA) 5 d postoperatively. Rats were immediately sacrificed and the hearts were excised. Atria and right ventricles were removed, and
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the left ventricles were weighed. Degree of hypertrophy was calculated in each coarctated and sham-operated rat using the ratio of left-ventricular weight (LVW) to body weight (BW). 2.4. Tissue homogenization and luciferase assay Left ventricles were divided into the left-ventricular apex (comprising the area of plasmid injection) and the leftventricular base. The ventricular apex tissues were homogenized in 500 µl of passive lysis buffer (Promega) per 100 mg of tissue and then centrifuged at 12,000 rpm for 20 min, yielding supernatant for the luciferase assay. A portion of the ventricular base was processed for histological staining and cell diameter measurement. The remainder of the tissue taken from rats at the 3 and 5-d post-operative time points was rapidly frozen in liquid nitrogen and processed for northern and western blot analyses. The firefly-luciferase activity of the SERCA2 test plasmid and the Renilla-luciferase activity of the pRL-CMV control plasmid were measured sequentially in a single tube using a Lumat LB 9507 luminometer (EG&G Berthold, Bad Wildbad, Germany). The transcriptional activity of the SERCA2 gene was determined as the ratio of light units of the SERCA2 promoter to pML-CMV control promoter. 2.5. RNA isolation and RNA blot analysis Total cellular RNA was isolated by a guanidine thiocyanate procedure as described previously [27]. Fifteen microgram of total RNA were fractionated on a 0.8% agarose gel, transferred to a filter and probed with various random-primed radiolabeled cDNA. The northern blot filters were washed with 0.5× SSC and 0.1% sodium dodecyl sulfate at 55 °C for 30 min. The level of expression of mRNA was normalized to that of GAPDH, an internal standard for RNA loading variations.
Fig. 2. Time course of SERCA2 transcriptional activity under pressure overload. Luciferase activities 3–14 d following DNA injection of the –284 to +350 bp SERCA2 promoter (no mutation)-luciferase reporter construct are illustrated. The transfection efficiency of the SERCA2 gene reporter vector was corrected by normalizing against the activity of CMV promoter connected to the sea pansy-luciferase reporter gene, which was transfected at the same time (dual-luciferase assay).
(Fig. 2). Luciferase activity in myocardial tissue homogenates at 3, 5, 7 and 14 d after injection of the –284 to +350 bp Luc (no mutation) plasmid were as follows: sham group (0.69 ± 0.15 at 3 d, 0.77 ± 0.01 at 5 d, 1.00 ± 0.36 at 7 d, 0.91 ± 0.20 at 14 d), pressure-overloaded group (0.60 ± 0.01 at 3 d, 0.39 ± 0.15 at 5 d, 0.45 ± 0.16 at 7 d, 0.39 ± 0.13 at 14 d). In contrast, transcription levels in the sham group were unchanged at all time points. Significant decreases in transcriptional activity were noted in the pressure-overloaded group at 5–14 d, when compared to the sham group. The LVW/BW ratio was significantly increased in the pressure-overloaded group at 5–14 d, when comparing to the sham group. Thus, all subsequent experiments were performed at 5 d postinjection.
2.6. Western blot analysis The ventricular base tissues were homogenized in sample buffer (10% SDS, 0.5 M Tris (pH 6.7), and 1 M DTT) and then centrifuged at 15,000 rpm for 10 min. The supernatants were used for western blot analysis. Ten microgram of protein were electrophoresed on 8% SDS-polyacrylamide gels and transferred onto a nitrocellulose membrane (Schleicher and Schuell, NH, USA). Sp1 protein was detected using a 1:100 dilution of a rabbit anti-Sp1 antibody (PEP-2, Santa Cruz Biotechnology, CA, USA).
3. Results 3.1. Time course of SERCA2 transcriptional activity after pressure overload Transcription of the reporter gene was quantified by assessing luciferase levels at various time points post-injection
3.2. Hemodynamic measurements in pressure-overloaded hearts To assess for cardiac hypertrophy, cardiac functional parameters were carefully monitored. Carotid systolic and diastolic pressures were significantly elevated in the pressureoverloaded group when compared to the control group (Table 1). There was no significant difference in presurgical BW between the two groups. However, LVW and LVW/BW ratio was significantly greater in the pressure-overloaded group, 5 d after surgery, when compared to control. Myocyte diameter was significantly greater in the pressure-overloaded group when compared to control. Absolute values of positive and negative dP/dt were significantly decreased in the pressure-overloaded group when compared to the control group, but no significant difference was found in LVEDP between the two groups. These data indicate induction of cardiac hypertrophy without congestive heart failure in the experimental group.
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Table 1 Hemodynamic measurements of pressure-overloaded hearts 5 d after surgery BW (g) Control (n = 46) 291 ± 21 Pressure overload 241 ± 25* (n = 47)
LVW (g) 0.67 ± 0.07 0.89 ± 0.10*
Cell diameter (µm) 19.39 ± 1.74
HR (bpm)
25.32 ± 1.68*
419 ± 48
SBP/DBP (mmHg) 141 ± 15/111 ±22 158 ± 20*/129 ± 26*
LVEDP (mmHg)
+dP/dt (mmHg/s) 4661 ± 411 3322 ± 393*
5.67 ± 2.92 8.51 ± 2.19
405 ± 45
LVW/BW (mg/g) 2.31 ± 0.17 3.71 ± 0.40*
–dP/dt (mmHg/s) 3850 ± 417 2621 ± 312*
BW, post-operation body weight; LVW, left-ventricular weight; HR, heart rate; SBP, systolic blood pressure; DBP, diastolic blood pressure; LVEDP, left-ventricular end-diastolic pressure. Values are means ± S.D. * Significantly different from control (P < 0.05).
3.3. Analysis of SERCA2 and Sp1 mRNA expression and Sp1 protein levels in pressure-overload cardiac hypertrophy SERCA2 mRNA levels in the pressure-overloaded group on day 5 were 75 ± 5% (P < 0.05) of control group levels. In contrast, Sp1 mRNA levels in the pressure-overloaded group
increased to 124 ± 7% of the control group levels (P < 0.05) (Fig. 3B,C), and Sp1 protein levels in the pressureoverloaded group increased to 160 ± 25% of the control group levels (P < 0.05) (Fig. 3B,C). To further clarify the relationship between Sp1 and SERCA2 gene expression, we examined changes in these parameters on day 3. As shown in Fig. 3A, SERCA2 mRNA levels and Sp1 mRNA and protein levels were similar when comparing control and pressureoverloaded group. These data suggest a role for Sp1 in the pressure-overload-induced suppression of SERCA2 mRNA expression (Fig. 3C).
3.4. Sp1 sites contribute to basal transcriptional activity of the SERCA2 gene
To determine the role of each Sp1 site in the transcriptional regulation of the SERCA2 gene, the luciferase reporter assay was performed for each Sp1 mutant and compared to the control promoter in control rat hearts (Fig. 4). Defining the transcriptional activity of the control group as 1.0, Sp1 mutation groups had the following relative activities: I = 0.45 ± 0.18; II = 0.51 ± 0.15; III = 0.18 ± 0.07; IV = 0.17 ± 0.05; I + II = 0.17 ± 0.06; I + II + III = 0.26 ± 0.06. Thus, SERCA2 transcription was significantly diminished in each of the Sp1 site mutation groups (P < 0.05).
Fig. 3. Expression of Sp1 mRNA and SERCA2 mRNA and Sp1 protein levels in pressure-overload cardiac hypertrophy. (A) Left panel (northern blot analysis). Expression of Sp1 mRNA and SERCA2 mRNA 3 d after aortic banding (pressure overload) or sham (control) operation is indicated in the upper two rows. The middle row displays expression of GAPDH mRNA obtained after stripping the same filter used for the Sp1 cDNA probe. The lower row displays an EtBr-stained gel. (A) Right panel (western blot analysis). The upper panel displays Sp1 proteins on day 3. The lower panel displays whole protein staining with Coomasie brilliant blue. Number of rats examined: control group = 8, pressure-overloaded (PO) group = 8. (B) Sp1 and SERCA2 mRNA expression and Sp1 protein measured 5 d after aortic banding (PO) or sham (control) operation are shown. Control group n = 7, pressure-overloaded (PO) group = 7. (C) Relative level of Sp1 mRNA, Sp1 protein and SERCA2 mRNA in the pressure-overloaded group compared with the control group on the respective post-operative days. Each parameter is expressed as a percentage of control values. Asterisk indicates a significant change in pressure-overload group when compared to the control group (P < 0.05).
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± 0.09. Therefore, pressure-overload-induced decreases in SERCA2 transcription under pressure overload were blocked by mutation of Sp1 site I (–196 to –191 bp site; P < 0.05) and III (–118 to –113 bp; P < 0.05). This suggests that Sp1 sites I and III, but not sites II or IV, are responsible for the inhibition of SERCA2 gene transcription under pressure overload. 4. Discussion
Fig. 4. Basal transcription of the SERCA2 gene (–284 to +350 bp) without pressure overload. Luciferase reporter gene assay was performed in each Sp1 mutant (I-IV, I + II, I + II + III). Sham wild-type and Sp1 mutant-luciferase reporter constructs were injected into the beating left-ventricular apex, and the luciferase activity was measured after 5 d and compared to the sham and Sp1 mutation groups. Measurements are expressed as a ratio of the sham group values. Values are expressed as means ± S.D. n = number of rats.
3.5. Sp1 sites I and III are responsible for inhibition of the SERCA2 gene under pressure overload In order to determine whether Sp1 sites contribute to pressure-overload-induced downregulation of gene transcription, hearts were injected with specific gene constructs, and luciferase assay was performed after 5 d of pressure overload (Fig. 5). Activity was expressed relative to the transcriptional activity of the sham-operated group. Pressure overload significantly reduced transcriptional activity of the wild-type promoter (spanning from –284 to +350 bp, i.e. no mutation group) to a relative ratio of 0.15 ± 0.04 (P < 0.05). The effect of each Sp1-site mutation on SERCA2 transcription in the pressure-overloaded cardiac hypertrophy was as follows: Sp1 site I = 0.54 ± 0.15; II = 0.25 ± 0.01; III = 0.56 ± 0.14; IV = 0.22 ± 0.07; I + II = 0.57 ± 0.09; I + II + III = 0.68
Fig. 5. Transcription of the SERCA2 gene (–284 to +350 bp) with pressureoverload cardiac hypertrophy. Hearts injected with indicated gene constructs (Fig. 1B) were exposed to pressure overload conditions for 5 d, and luciferase activity was measured in each construct. The transcriptional activity of the hypertrophy group was measured and compared to the transcriptional activity of the sham group (defined as 1.0). Values are expressed as means ± S.D.* P < 0.05. n = number of rats.
Previous reports have demonstrated decreased SERCA2 mRNA and protein levels with pressure-overload cardiac hypertrophy [8,25,26]. Recent studies have utilized an in vivo direct gene transfer method to demonstrate that inhibition of SERCA2 gene transcription was responsible for decreased SERCA2 mRNA levels in pressure-overload states. Further, two putative transcription regulatory domains (–1810 to –1110 and –284 to –72 bp regions) were identified [8,16,28]. The present study characterized the role of the proximal promoter in SERCA2 gene transcription and describes the contribution of the Sp1 transcription factor in pressure-overload-induced changes in the SERCA2 gene transcriptional activity. We demonstrated that SERCA2 gene transcription significantly decreased 5 d after exposure to pressure overload and remained lower than sham group thereafter. Putative-binding sites for GATA-1, CArG, CRE, and MCAT (–1810 to –1110 bp region) and for AP2, Sp1, and Egr-1 (–284 to –72 bp region) are present in the SERCA2 gene promoter. Muscle-specific genes, including cardiac and skeletal a-actin [14,15], b-myosin heavy chain [13], and SERCA2 [16], require Sp1 for efficient gene expression. Further, a1-adrenergic-mediated induction of the skeletal a-actin and ANP gene is dependent on Sp1 and CArG elements [17,18]. The SERCA2 gene has a cluster of putative Sp1-binding sites in the proximal region of the promoter, making Sp1-binding sites attractive candidates for mediation of pressure-overload-induced SERCA2 transcription downregulation. Consistent with this hypothesis, mRNA and protein levels of Sp1 were elevated in pressure-overloaded hearts (Fig. 3). Mutation of Sp1 sites I and III, but not sites II or IV, blocked pressure-overload-induced decreases in transcription (Fig. 5). We have previously demonstrated that Sp1 sites I and III are essential for muscle-specific SERCA2 expression in a slow-twitch skeletal muscle cell line (Sol 8) [16], establishing the importance of Sp1 sites I and III in SERCA2 transcriptional regulation in muscle cells. In contrast, the present data demonstrated that all four Sp1 sites contribute to the basal transcriptional activity of the SERCA2 gene in cardiac myocytes (Fig. 4). These contrasting findings may be due to differences in cell types or secondary to differences between cultured cell lines and tissue in vivo. Interestingly, Sp1 sites also mediated pressure-overloadinduced decreases in SERCA2 gene expression. The precise mechanism of the dual role of Sp1 sites is not clear; however, the ratio of Sp1 transcription factors may be an important
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determinant of the positive or negative transcriptional effect. The GC-rich sequence (which is defined as the Sp1 site in this study) has similar binding affinities as other Sp family transcription factors, such as Sp3 and Sp4 [29,30]. GC-rich elements are essential for both basal and hypoxia-induced activation of the muscle-specific pyruvate kinase-M and b-enolase genes [31]. Both Sp1 and Sp3 bind to the GC-rich element of these genes. Hypoxia decreases binding of Sp3 and increases DNA-bound Sp1, thereby enhancing transcription. In another example, the ornithine decarboxylase gene is positively regulated by Sp1 and negatively regulated by Sp3 during hepatocyte development and cellular transformation by oncogenes [32]. Although we did not study Sp1 and Sp3 binding in this model, it is likely that transcriptional regulation follows schemes similar to those already reported. Investigators have recently characterized the role of cisand trans-acting factors in cardiac hypertrophy-mediated muscle-specific gene regulation. Wright et al. [33] demonstrated that three regions in the b-myosin heavy chain gene promoter responded to abdominal aortic banding in rats, but failed to isolate the specific transcription factor. Hautala et al. [34] described arginine vasopressin-induced brain natriuretic peptide gene transcription mediated by GATA4. These studies utilized cultured neonatal cardiac myocyte systems that lack the influence of the nervous system and circulating hormones. The present study identifies two Sp1-binding sites as transcriptional regulatory elements in the SERCA2 gene. In contrast to previous studies, the use of reporter genes in an in vivo model has the advantage of closely approximating normal physiology. In conclusion, this study demonstrated that Sp1 sites in the proximal region of the SERCA2 promoter mediate basal and pressure-overload-induced changes in transcriptional activity. These findings may have implications for the treatment of patients with cardiac hypertrophy and secondary heart failure.
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Original Article
Transcription factor Sp1 regulates SERCA2 gene expression in pressure-overloaded hearts: a study using in vivo direct gene transfer into living myocardium > Takako Takizawa a, Masashi Arai a,*, Koichi Tomaru a, Norimichi Koitabashi a, Debra L. Baker b, Muthu Periasamy b, Masahiko Kurabayashi a a b
Second Department of Internal Medicine, Gunma University School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma 371-8511, Japan Department of Physiology and Cell Biology, College of Medicine and Health Sciences, Ohio State University, Columbus, OH 43210-1218, USA Received 24 February 2003; accepted 19 March 2003
Abstract Pressure-overload hypertrophy results in downregulation of the sarcoplasmic reticulum Ca2+-ATPase pump encoding SERCA2 gene that regulates Ca2+ uptake and myocardial relaxation. We previously characterized a proximal promoter region containing four Sp1 element consensus sequences (–284 to –72 base pairs (bp)) that was responsible for pressure-overload-induced transcriptional regulation. The purpose of the present study was to determine which of the Sp1 sites was responsible for the downregulation of SERCA2 gene transcription under pressure overload. Using an in vivo direct gene transfer assay, SERCA2 gene transcriptional activity was measured under pressure overload. Site-directed mutagenesis of the four Sp1 sites (I-IV) in the SERCA2 gene promoter (–284 to –72 bp) was performed. Wild-type and Sp1 mutant-luciferase reporter constructs were injected into the left-ventricular apices of pressure overload or sham-operated rats, and Sp1 mRNA and SERCA2 gene-luciferase activity was measured sequentially from 3 to 14 d after surgery. At 5 d, Sp1 mRNA in the pressure-overload rats increased to 124 ± 7% of sham group levels, and pressure-overload-induced SERCA2 transcriptional activity was 15 ± 4% of sham group when all four Sp1 sites remained intact. Mutation of the Sp1 mutant sites I (–196 to –191 bp) and III (–118 to –113 bp) blocked the inhibitory effect of pressure overload and resulted in SERCA2 gene transcriptional activity of 54 ± 15% and 56 ± 7% of sham group, respectively. We conclude that the pressure-overload-induced decrease in SERCA2 mRNA is mediated by Sp1 sites I and III. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: SERCA2; Pressure overload; Cardiac hypertrophy; Sp1; Gene transcription factor; Luciferase assay; In vivo gene transfer
1. Introduction The sarcoplasmic reticulum (SR) plays an important role in the cardiac contraction-relaxation cycle. Myocardial contraction is triggered by Ca2+ release from the SR via ryanodine receptors, and reuptake of Ca2+ via the SR Ca2+-ATPase (SERCA2) pump results in relaxation. Previous reports suggested that pressure overload results in decreases in SERCA2 mRNA, SERCA2 protein concentration, and Ca2+ reuptake function [1–7]. We recently demonstrated that decreases in SERCA2 mRNA levels resulted from downregulation of
> The review of this manuscript was handled by consulting editor, Stephen F. Vatner, M.D. * Corresponding author. Tel.: +81-27-220-8145; fax: +81-27-220-8158. E-mail address: [email protected] (M. Arai).
© 2003 Elsevier Science Ltd. All rights reserved. DOI: 10.1016/S0022-2828(03)00122-6
SERCA2 gene transcription [8] via regulatory sites in the SERCA2 gene promoter (distal site, –1810 to –1110 base pairs (bp); proximal site, –284 to –72 bp). Pressure overload results in altered transcription profiles of various genes. Genes affected by pressure overload include atrial natriuretic peptide (ANP), the angiotensin II type-1a receptor and myocardial lipid metabolism (MCLM) genes, which are under control of transcription factors such as AP-1, CRE, GATA-4, Sp1, and Sp3 [9–12]. While the SERCA2 gene lacks binding sites for AP-1 and Sp3, CRE, and GATA-binding sites are present between –1810 and –1110 bp, and four Sp1 sites are present in the proximal region between –284 and –72 bp. However, the contribution of each individual site in SERCA2 gene transcription regulation during pressure-overload-induced hypertrophy has not been characterized.
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Muscle-specific genes, including cardiac a-actin, skeletal a-actin, b-myosin heavy chain, and SERCA2, require Sp1 for efficient gene expression [13–16]. Further, a1-adrenergic-mediated induction of the skeletal a-actin and ANP genes is dependent on Sp1 and CArG elements [17,18]. As Sp1 appears to play an essential role in the regulation of musclespecific genes, the present study investigated the importance of the proximal regulatory region that contains four putative Sp1-binding sites to SERCA2 gene transcription and attempted to characterize the contribution of each individual Sp1-binding site. By injecting reporter genes into beating hearts, we characterized the role of each individual Sp1binding site on SERCA2 gene transcription in an in vivo model of cardiac hypertrophy.
2. Materials and methods 2.1. DNA probes The following cDNA fragments were used as mRNA hybridization probes: 1. rabbit SR Ca2+-ATPase2 (SERCA2) cDNA [19], nucleotides 1798–3594; 2. rat Sp1 cDNA [20,21], nucleotides 21–925; 3. human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA [22], nucleotides 37–740. 2.2. Reporter gene construction The proximal promoter region of the SERCA2 gene (–284 to –72 bp) contains four putative Sp1-binding sites (Fig. 1A). Mutants were generated using the altered sites II in vitro mutagenesis kit (Promega) to eliminate Sp1-binding sites. Second and third guanine or cytosine nucleotides were replaced with two thymidine nucleotides in each Sp1 consensus sequence (Fig. 1A). Four single mutation (I-IV), double mutation (I + II) and triple mutation (I + II + III) in Sp1 sites were created within the SERCA2 promoter region from –284 to +350 bp relative to the transcription start site and connected to the fireflyluciferase reporter gene. Transcriptional activity of the SERCA2 gene was determined in the various mutant groups and compared with wild-type promoter construct (Fig. 1B). Control vectors containing the CMV promoter (pRL-CMV sea pansy luciferase) were obtained from a commercial source (Promega, Madison, WI, USA) and used for correction of transfection efficiency variation. 2.3. Direct plasmid injection into in vivo myocardium and hemodynamic measurements Plasmids were directly injected into the myocardium of adult Wistar rats as previously described [23,24]. Briefly, 50 µl of normal saline solution containing 10 µg of SERCA2 firefly-luciferase plasmid and 1 µg of pRL-CMV control plasmid were injected via a 30-G needle into the left-
Fig. 1. (A) Nucleotide sequence of the SERCA2 proximal promoter (–284 to –72 bp). The consensus binding sequences for the Sp1 transcription factor (sites I-VI) are indicated. Second and third guanine or cytosine nucleotides were replaced with two thymine nucleotides in each Sp1 consensus sequence by using in vitro site-specific mutagenesis. (B) Plasmid construction. Schematic representation of mutations in individual Sp1 consensus sites (I-VI) and in combinations (I + II and I + II + III). The Sp1 consensus sequences were mutated from consensus GGGCGG to GTTCGG by sitedirected mutagenesis of the –284 to +350 bp SERCA2 promoter. Six constructs of the SERCA2 promoter-luciferase reporter plasmid were used.
ventricular apex. The activities of firefly luciferase and sea pansy luciferase were measured sequentially from a single sample (dual-luciferase reporter assay system, Promega, Madison, WI, USA). The titer of firefly luciferase representing the activity of SERCA2 gene promoter was normalized to sea pansy luciferase activity, an internal standard of transfection efficiency. Abdominal aortic constriction was achieved concomitant to plasmid injection via a silver aortic clip placed between the renal and superior mesenteric arteries [8,25,26]. Sham-operated controls underwent the same processing as experimental animals except for placement of the aortic clip. To determine the time course of transcriptional activity of the SERCA2 gene after pressure overload, rats were sacrificed at various time points (3, 5, 7 and 14 d post-operatively, n = 4 for each time point). Measurements of ascending aortic pressure, leftventricular end-diastolic pressure (LVEDP), dP/dt, and heart rate were performed via the carotid artery using a Millar catheter (3Fr Millar Instruments, Inc., USA) 5 d postoperatively. Rats were immediately sacrificed and the hearts were excised. Atria and right ventricles were removed, and
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the left ventricles were weighed. Degree of hypertrophy was calculated in each coarctated and sham-operated rat using the ratio of left-ventricular weight (LVW) to body weight (BW). 2.4. Tissue homogenization and luciferase assay Left ventricles were divided into the left-ventricular apex (comprising the area of plasmid injection) and the leftventricular base. The ventricular apex tissues were homogenized in 500 µl of passive lysis buffer (Promega) per 100 mg of tissue and then centrifuged at 12,000 rpm for 20 min, yielding supernatant for the luciferase assay. A portion of the ventricular base was processed for histological staining and cell diameter measurement. The remainder of the tissue taken from rats at the 3 and 5-d post-operative time points was rapidly frozen in liquid nitrogen and processed for northern and western blot analyses. The firefly-luciferase activity of the SERCA2 test plasmid and the Renilla-luciferase activity of the pRL-CMV control plasmid were measured sequentially in a single tube using a Lumat LB 9507 luminometer (EG&G Berthold, Bad Wildbad, Germany). The transcriptional activity of the SERCA2 gene was determined as the ratio of light units of the SERCA2 promoter to pML-CMV control promoter. 2.5. RNA isolation and RNA blot analysis Total cellular RNA was isolated by a guanidine thiocyanate procedure as described previously [27]. Fifteen microgram of total RNA were fractionated on a 0.8% agarose gel, transferred to a filter and probed with various random-primed radiolabeled cDNA. The northern blot filters were washed with 0.5× SSC and 0.1% sodium dodecyl sulfate at 55 °C for 30 min. The level of expression of mRNA was normalized to that of GAPDH, an internal standard for RNA loading variations.
Fig. 2. Time course of SERCA2 transcriptional activity under pressure overload. Luciferase activities 3–14 d following DNA injection of the –284 to +350 bp SERCA2 promoter (no mutation)-luciferase reporter construct are illustrated. The transfection efficiency of the SERCA2 gene reporter vector was corrected by normalizing against the activity of CMV promoter connected to the sea pansy-luciferase reporter gene, which was transfected at the same time (dual-luciferase assay).
(Fig. 2). Luciferase activity in myocardial tissue homogenates at 3, 5, 7 and 14 d after injection of the –284 to +350 bp Luc (no mutation) plasmid were as follows: sham group (0.69 ± 0.15 at 3 d, 0.77 ± 0.01 at 5 d, 1.00 ± 0.36 at 7 d, 0.91 ± 0.20 at 14 d), pressure-overloaded group (0.60 ± 0.01 at 3 d, 0.39 ± 0.15 at 5 d, 0.45 ± 0.16 at 7 d, 0.39 ± 0.13 at 14 d). In contrast, transcription levels in the sham group were unchanged at all time points. Significant decreases in transcriptional activity were noted in the pressure-overloaded group at 5–14 d, when compared to the sham group. The LVW/BW ratio was significantly increased in the pressure-overloaded group at 5–14 d, when comparing to the sham group. Thus, all subsequent experiments were performed at 5 d postinjection.
2.6. Western blot analysis The ventricular base tissues were homogenized in sample buffer (10% SDS, 0.5 M Tris (pH 6.7), and 1 M DTT) and then centrifuged at 15,000 rpm for 10 min. The supernatants were used for western blot analysis. Ten microgram of protein were electrophoresed on 8% SDS-polyacrylamide gels and transferred onto a nitrocellulose membrane (Schleicher and Schuell, NH, USA). Sp1 protein was detected using a 1:100 dilution of a rabbit anti-Sp1 antibody (PEP-2, Santa Cruz Biotechnology, CA, USA).
3. Results 3.1. Time course of SERCA2 transcriptional activity after pressure overload Transcription of the reporter gene was quantified by assessing luciferase levels at various time points post-injection
3.2. Hemodynamic measurements in pressure-overloaded hearts To assess for cardiac hypertrophy, cardiac functional parameters were carefully monitored. Carotid systolic and diastolic pressures were significantly elevated in the pressureoverloaded group when compared to the control group (Table 1). There was no significant difference in presurgical BW between the two groups. However, LVW and LVW/BW ratio was significantly greater in the pressure-overloaded group, 5 d after surgery, when compared to control. Myocyte diameter was significantly greater in the pressure-overloaded group when compared to control. Absolute values of positive and negative dP/dt were significantly decreased in the pressure-overloaded group when compared to the control group, but no significant difference was found in LVEDP between the two groups. These data indicate induction of cardiac hypertrophy without congestive heart failure in the experimental group.
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Table 1 Hemodynamic measurements of pressure-overloaded hearts 5 d after surgery BW (g) Control (n = 46) 291 ± 21 Pressure overload 241 ± 25* (n = 47)
LVW (g) 0.67 ± 0.07 0.89 ± 0.10*
Cell diameter (µm) 19.39 ± 1.74
HR (bpm)
25.32 ± 1.68*
419 ± 48
SBP/DBP (mmHg) 141 ± 15/111 ±22 158 ± 20*/129 ± 26*
LVEDP (mmHg)
+dP/dt (mmHg/s) 4661 ± 411 3322 ± 393*
5.67 ± 2.92 8.51 ± 2.19
405 ± 45
LVW/BW (mg/g) 2.31 ± 0.17 3.71 ± 0.40*
–dP/dt (mmHg/s) 3850 ± 417 2621 ± 312*
BW, post-operation body weight; LVW, left-ventricular weight; HR, heart rate; SBP, systolic blood pressure; DBP, diastolic blood pressure; LVEDP, left-ventricular end-diastolic pressure. Values are means ± S.D. * Significantly different from control (P < 0.05).
3.3. Analysis of SERCA2 and Sp1 mRNA expression and Sp1 protein levels in pressure-overload cardiac hypertrophy SERCA2 mRNA levels in the pressure-overloaded group on day 5 were 75 ± 5% (P < 0.05) of control group levels. In contrast, Sp1 mRNA levels in the pressure-overloaded group
increased to 124 ± 7% of the control group levels (P < 0.05) (Fig. 3B,C), and Sp1 protein levels in the pressureoverloaded group increased to 160 ± 25% of the control group levels (P < 0.05) (Fig. 3B,C). To further clarify the relationship between Sp1 and SERCA2 gene expression, we examined changes in these parameters on day 3. As shown in Fig. 3A, SERCA2 mRNA levels and Sp1 mRNA and protein levels were similar when comparing control and pressureoverloaded group. These data suggest a role for Sp1 in the pressure-overload-induced suppression of SERCA2 mRNA expression (Fig. 3C).
3.4. Sp1 sites contribute to basal transcriptional activity of the SERCA2 gene
To determine the role of each Sp1 site in the transcriptional regulation of the SERCA2 gene, the luciferase reporter assay was performed for each Sp1 mutant and compared to the control promoter in control rat hearts (Fig. 4). Defining the transcriptional activity of the control group as 1.0, Sp1 mutation groups had the following relative activities: I = 0.45 ± 0.18; II = 0.51 ± 0.15; III = 0.18 ± 0.07; IV = 0.17 ± 0.05; I + II = 0.17 ± 0.06; I + II + III = 0.26 ± 0.06. Thus, SERCA2 transcription was significantly diminished in each of the Sp1 site mutation groups (P < 0.05).
Fig. 3. Expression of Sp1 mRNA and SERCA2 mRNA and Sp1 protein levels in pressure-overload cardiac hypertrophy. (A) Left panel (northern blot analysis). Expression of Sp1 mRNA and SERCA2 mRNA 3 d after aortic banding (pressure overload) or sham (control) operation is indicated in the upper two rows. The middle row displays expression of GAPDH mRNA obtained after stripping the same filter used for the Sp1 cDNA probe. The lower row displays an EtBr-stained gel. (A) Right panel (western blot analysis). The upper panel displays Sp1 proteins on day 3. The lower panel displays whole protein staining with Coomasie brilliant blue. Number of rats examined: control group = 8, pressure-overloaded (PO) group = 8. (B) Sp1 and SERCA2 mRNA expression and Sp1 protein measured 5 d after aortic banding (PO) or sham (control) operation are shown. Control group n = 7, pressure-overloaded (PO) group = 7. (C) Relative level of Sp1 mRNA, Sp1 protein and SERCA2 mRNA in the pressure-overloaded group compared with the control group on the respective post-operative days. Each parameter is expressed as a percentage of control values. Asterisk indicates a significant change in pressure-overload group when compared to the control group (P < 0.05).
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± 0.09. Therefore, pressure-overload-induced decreases in SERCA2 transcription under pressure overload were blocked by mutation of Sp1 site I (–196 to –191 bp site; P < 0.05) and III (–118 to –113 bp; P < 0.05). This suggests that Sp1 sites I and III, but not sites II or IV, are responsible for the inhibition of SERCA2 gene transcription under pressure overload. 4. Discussion
Fig. 4. Basal transcription of the SERCA2 gene (–284 to +350 bp) without pressure overload. Luciferase reporter gene assay was performed in each Sp1 mutant (I-IV, I + II, I + II + III). Sham wild-type and Sp1 mutant-luciferase reporter constructs were injected into the beating left-ventricular apex, and the luciferase activity was measured after 5 d and compared to the sham and Sp1 mutation groups. Measurements are expressed as a ratio of the sham group values. Values are expressed as means ± S.D. n = number of rats.
3.5. Sp1 sites I and III are responsible for inhibition of the SERCA2 gene under pressure overload In order to determine whether Sp1 sites contribute to pressure-overload-induced downregulation of gene transcription, hearts were injected with specific gene constructs, and luciferase assay was performed after 5 d of pressure overload (Fig. 5). Activity was expressed relative to the transcriptional activity of the sham-operated group. Pressure overload significantly reduced transcriptional activity of the wild-type promoter (spanning from –284 to +350 bp, i.e. no mutation group) to a relative ratio of 0.15 ± 0.04 (P < 0.05). The effect of each Sp1-site mutation on SERCA2 transcription in the pressure-overloaded cardiac hypertrophy was as follows: Sp1 site I = 0.54 ± 0.15; II = 0.25 ± 0.01; III = 0.56 ± 0.14; IV = 0.22 ± 0.07; I + II = 0.57 ± 0.09; I + II + III = 0.68
Fig. 5. Transcription of the SERCA2 gene (–284 to +350 bp) with pressureoverload cardiac hypertrophy. Hearts injected with indicated gene constructs (Fig. 1B) were exposed to pressure overload conditions for 5 d, and luciferase activity was measured in each construct. The transcriptional activity of the hypertrophy group was measured and compared to the transcriptional activity of the sham group (defined as 1.0). Values are expressed as means ± S.D.* P < 0.05. n = number of rats.
Previous reports have demonstrated decreased SERCA2 mRNA and protein levels with pressure-overload cardiac hypertrophy [8,25,26]. Recent studies have utilized an in vivo direct gene transfer method to demonstrate that inhibition of SERCA2 gene transcription was responsible for decreased SERCA2 mRNA levels in pressure-overload states. Further, two putative transcription regulatory domains (–1810 to –1110 and –284 to –72 bp regions) were identified [8,16,28]. The present study characterized the role of the proximal promoter in SERCA2 gene transcription and describes the contribution of the Sp1 transcription factor in pressure-overload-induced changes in the SERCA2 gene transcriptional activity. We demonstrated that SERCA2 gene transcription significantly decreased 5 d after exposure to pressure overload and remained lower than sham group thereafter. Putative-binding sites for GATA-1, CArG, CRE, and MCAT (–1810 to –1110 bp region) and for AP2, Sp1, and Egr-1 (–284 to –72 bp region) are present in the SERCA2 gene promoter. Muscle-specific genes, including cardiac and skeletal a-actin [14,15], b-myosin heavy chain [13], and SERCA2 [16], require Sp1 for efficient gene expression. Further, a1-adrenergic-mediated induction of the skeletal a-actin and ANP gene is dependent on Sp1 and CArG elements [17,18]. The SERCA2 gene has a cluster of putative Sp1-binding sites in the proximal region of the promoter, making Sp1-binding sites attractive candidates for mediation of pressure-overload-induced SERCA2 transcription downregulation. Consistent with this hypothesis, mRNA and protein levels of Sp1 were elevated in pressure-overloaded hearts (Fig. 3). Mutation of Sp1 sites I and III, but not sites II or IV, blocked pressure-overload-induced decreases in transcription (Fig. 5). We have previously demonstrated that Sp1 sites I and III are essential for muscle-specific SERCA2 expression in a slow-twitch skeletal muscle cell line (Sol 8) [16], establishing the importance of Sp1 sites I and III in SERCA2 transcriptional regulation in muscle cells. In contrast, the present data demonstrated that all four Sp1 sites contribute to the basal transcriptional activity of the SERCA2 gene in cardiac myocytes (Fig. 4). These contrasting findings may be due to differences in cell types or secondary to differences between cultured cell lines and tissue in vivo. Interestingly, Sp1 sites also mediated pressure-overloadinduced decreases in SERCA2 gene expression. The precise mechanism of the dual role of Sp1 sites is not clear; however, the ratio of Sp1 transcription factors may be an important
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determinant of the positive or negative transcriptional effect. The GC-rich sequence (which is defined as the Sp1 site in this study) has similar binding affinities as other Sp family transcription factors, such as Sp3 and Sp4 [29,30]. GC-rich elements are essential for both basal and hypoxia-induced activation of the muscle-specific pyruvate kinase-M and b-enolase genes [31]. Both Sp1 and Sp3 bind to the GC-rich element of these genes. Hypoxia decreases binding of Sp3 and increases DNA-bound Sp1, thereby enhancing transcription. In another example, the ornithine decarboxylase gene is positively regulated by Sp1 and negatively regulated by Sp3 during hepatocyte development and cellular transformation by oncogenes [32]. Although we did not study Sp1 and Sp3 binding in this model, it is likely that transcriptional regulation follows schemes similar to those already reported. Investigators have recently characterized the role of cisand trans-acting factors in cardiac hypertrophy-mediated muscle-specific gene regulation. Wright et al. [33] demonstrated that three regions in the b-myosin heavy chain gene promoter responded to abdominal aortic banding in rats, but failed to isolate the specific transcription factor. Hautala et al. [34] described arginine vasopressin-induced brain natriuretic peptide gene transcription mediated by GATA4. These studies utilized cultured neonatal cardiac myocyte systems that lack the influence of the nervous system and circulating hormones. The present study identifies two Sp1-binding sites as transcriptional regulatory elements in the SERCA2 gene. In contrast to previous studies, the use of reporter genes in an in vivo model has the advantage of closely approximating normal physiology. In conclusion, this study demonstrated that Sp1 sites in the proximal region of the SERCA2 promoter mediate basal and pressure-overload-induced changes in transcriptional activity. These findings may have implications for the treatment of patients with cardiac hypertrophy and secondary heart failure.
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