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Cardiovascular Research 1998 37(2):503-514; doi:10.1016/S0008-6363(97)00254-X
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Sarcoplasmic reticulum Ca2+ ATPase promoter activity during endothelin-1 induced hypertrophy of cultured rat cardiomyocytes

Han A.A. van Heugtena, Marga C. van Settena, Karin Eizemaa, Pieter D. Verdouwb and Jos M.J. Lamersa,*

aDepartment of Biochemistry, Cardiovascular Research Institute COEUR, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, Netherlands
bThorax Centre, Experimental Cardiology, Cardiovascular Research Institute COEUR, Erasmus University Rotterdam, 3000 DR Rotterdam, Netherlands

* Corresponding author. Tel.: +31 (10) 4087335; fax: +31 (10) 4360615.

Received 5 August 1997; accepted 17 October 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: Characterization of an in vitro model of endothelin-1 induced hypertrophy of cultured neonatal rat ventricular myocytes and subsequent analysis of transcription regulation of the rat promoter of the sarcoplasmic reticulum Ca2+ ATPase gene. Methods: Neonatal rat ventricular myocytes were cultured in serum free medium and hypertrophy was induced by addition of endothelin-1 to 10–8 M up to 48 h. Hypertrophy was characterized biochemically, and gene expression regulation was evaluated by Northern blotting. A sarcoplasmic reticulum Ca2+ ATPase promoter fragment, isolated from a rat library was cloned in a reporter vector. Promoter activity during hypertrophy was assessed after transfection of the reporter plasmid to cultured cardiomyocytes. Results: Stimulation with endothelin-1 resulted in increased cell size, as indicated by protein/DNA ratio as well as by augmented protein synthesis. When compared to angiotensin II or {alpha}1-adrenergic agonist, endothelin-1 was the strongest inducer of hypertrophy (protein/DNA ratio) after 48 h of stimulation. Endothelin-1 induced hypertrophy was accompanied by a twofold increase in total RNA content per cell as well as to increased glyceraldehydephosphate dehydrogenase mRNA levels. The level of atrial natriuretic factor mRNA was increased more than twofold, relative to glyceraldehydephosphate dehydrogenase, while the expression of the sarcoplasmic reticulum Ca2+ pump and phospholamban genes was decreased (by 26 and 49%, respectively) after induction of hypertrophy by stimulation with endothelin-1. In the same model, a 1.9 kb sarcoplasmic reticulum Ca2+ pump gene promoter fragment (including 0.4 kb of the 5' UTR of the mRNA) directed down-regulation of the expression of the reporter gene to the same magnitude as endogenous Ca2+ pump mRNA relative to glyceraldehydephosphate dehydrogenase mRNA. However, absolute mRNA level per cell did not change for either the reporter gene or the endogenous Ca2+ pump. Conclusions: Endothelin-1 can induce phenotypic changes in cultured rat ventricular myocytes that are reminiscent of hypertrophy in vivo. In this model, a 1.9 kb sarcoplasmic reticulum Ca2+ pump promoter fragment directed gene expression of a reporter gene identical to the endogenous regulation of the Ca2+ pump. Furthermore, expression of the Ca2+ pump during hypertrophy was only downregulated when compared to (increased levels of) glyceraldehydephosphate dehydrogenase mRNA, but absolute Ca2+ ATPase mRNA amounts remained unchanged. This suggests that the Ca2+ pump promoter is not responding to the increase in transcriptional activity that accompanies hypertrophy.

KEYWORDS Ventricular myocytes; Hypertrophy; Endothelin-1; Sarcoplasmic reticulum Ca2+ ATPase; Gene expression; Promoter activity; Rat


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Growth of the embryonic heart occurs through controlled mitotic division of myocytes. After birth, this proliferative capacity of myocytes rapidly decreases to a very low level. As a result, adaptation of the heart to increased workload after myocardial infarction or during chronic hypertension can mainly take place by an increase in size of individual cardiac myocytes called hypertrophy. However, excessive hypertrophy is accompanied by changes in gene expression that can be summarized as a reversal to a fetal phenotype [1]. These changes include (i) induction of gene transcription e.g. for atrial natriuretic factor (ANF), (ii) changes in isozyme expression, e.g. the shift in myosin heavy chain (MHC) expression from the {alpha}- to the β-form in rodents, (iii) upregulation of gene expression as was demonstrated for the Na+–Ca2+ exchanger, and (iv) downregulation of gene expression as is the case for e.g. the sarcoplasmic reticulum (SR) Ca2+-ATPase (Ca2+-pump) and phospholamban genes. Although Ca2+-pump expression during hypertrophy is decreased, regulation at the protein level is still controversial [2–4]. However, it is well documented that Ca2+-pump activity is decreased during hypertrophy [5]. Some of the changes in transcription described above can be judged to be compensatory for the increased workload posed on the heart, like induction of the vasodilator ANF (reduction of hemodynamic load) or expression of β-MHC that exhibits a lower velocity of ATP-cycling (improved economy). On the other hand, the decreased Ca2+-pump activity will lead to decreased clearing of Ca2+ from the sarcoplasm resulting in impairment of diastolic dysfunction as was also described before for cultured cardiac myocytes [6]. This might be one of the changes that ultimately leads to the development of heart failure, the inevitable outcome of prolonged hypertrophy [7].

Results from in vitro studies show that a large number of factors can induce hypertrophy of cultured cardiomyocytes, including e.g. {alpha}1-adrenergic agonists, endothelin-1 (ET-1), angiotensin II (AngII), growth factors or stretch (see e.g. [8]and references therein). In several animal models of hypertrophy induction it has been shown that both ET-1 and AngII play a prominent role in this process in vivo [9–12], suggesting a multifactorial origin for development of hypertrophy.

To study transcriptional regulation of the cardiac Ca2+-pump gene SERCA2 during hypertrophy, we employed a model of cultured neonatal rat ventricular myocytes. This model allows analysis of hypertrophy, functional characterization of the Ca2+-pump, transfection of foreign DNA and analysis of gene expression. We isolated the promoter of the SERCA2 gene from a rat genomic library, and show in this study that a 1.56 kb 5' upstream regulatory region together with 0.36 kb 5' UTR of this gene directs transcriptional regulation upon stimulation with ET-1 analogous to the endogenous SERCA2 gene in cardiomyocytes. We will discuss, in comparison to the general increase in transcriptional activity, the observed decrease in SERCA2 mRNA expression in the light of the possible strength of the TATA box(es) present in this promoter.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Reagents
Culture dishes (Falcon 3004) were obtained from Becton Dickinson (Plymouth, England). Culture media DMEM and M199 were from GibcoBRL (Life Technologies, Scotland), while fetal calf and horse sera were from Boehringer Mannheim (Germany). Endothelin-1 was obtained from Peninsula Laboratories (Belmont CA, USA). L-[4,5-3H]leucine (54 Ci/mmol) and [5,6-3H]uridine (41 Ci/mmol) were from Amersham (UK).

2.2 Library screening for isolation of a large rat SERCA2 5' regulatory region
All molecular biological techniques were performed according to standard protocols [13], unless indicated otherwise. For screening, a rat genomic library (Stratagene) was used. This library was constructed in Lambda DASHII and contained 2.0·106 independent clones with an insert size of 9–22 kb. Roughly 106 recombinant phages were plated on 20 plates and blotted to Hybond-N (Amersham). A first screening was performed with a random-prime (Megaprime, Amersham) labelled SERCA2 cDNA fragment (830 bp; an exon 4–exon 9 containing EcoRI/PstI fragment) according to protocols of the supplier. Briefly, filters were prehybridized in 20 mM Piperazine-N,N'-bis(2-ethanesulfonic acid) (pH 6.5), 0.8 M NaCl, 50% formamide, 0.5% SDS and 0.1 mg/ml denatured fish DNA (2 ml/filter) for 4 h at 42°C. Thereafter, labelled probe (107 cpm/filter) was added and hybridization was continued overnight. Subsequently, filters were washed for 5 min with 2xSSC, 0.1% SDS at room temperature followed by two washes for 15 min at 60°C with 0.1xSSC, 0.1% SDS. This primary screening yielded 30 positive plaques. A secondary screening (in 5 pooled groups of 6 plaques) was performed at lower stringency (3 washes with 2xSSC, 0.1% SDS at room temperature for 20 min each) with a short promoter-specific probe. This probe was generated by PCR with primers based on a published 559 bp rat promoter fragment yielding a 341 bp fragment extending from to –353 to –12 relative to the transcription start site [14]. This secondary screening yielded 3 positive plaques, of which two arose from the same plate. After isolation of DNA from these phages, restriction analysis with EcoRI, BamHI and HindIII followed by Southern blotting with the promoter probe showed that these three clones were identical and contained unique promoter-probe positive fragments for each restriction enzyme (results not shown).

2.3 Characterization of the SERCA2 promoter and construction of expression vectors
After large scale phage preparation, isolated phage DNA with SERCA2-insert was subjected to restriction mapping and Southern blotting with the promoter probe. Restriction mapping was performed with the FLASH gene mapping kit (Stratagene) according to the protocol of the supplier. In short, the insert was excised from the Lambda DASH II vector with SalI, leaving intact T3 and T7 promoter sites on either side of the insert. After generation of partial digests by enzyme dilution with single restriction enzymes, fragments were separated on a 10 cm long 0.8% agarose gel, denatured for 45 min in 1.5 M NaCl, 0.5 M NaOH, neutralised for 30 min in 1.5 M NaCl, 1 M Tris/HCl (pH 8) and blotted onto Hybond. After hybridisation with either T3 or T7 alkaline phosphatase-conjugated probe and development of the chemiluminescent CSPD substrate signal by exposure to X-ray film, successive restriction sites could be calculated from the size of the ladder of detected bands. This resulted in the partial restriction map displayed in Fig. 1 (Top). In a separate Southern blotting experiment, the SERCA2 insert was excised with SalI, and subsequently completely digested with the restriction enzymes depicted in Fig. 1 (Top), blotted and probed with either the promoter probe or the cDNA fragment (exon 4–9) described above. The minimal region that was positive for either of these probes is depicted in Fig. 1 as well (large arrow and box respectively).


Figure 1
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  		Fig. 1 Restriction map of a genomic rat SERCA2 gene fragment. A genomic clone for the SERCA2 was isolated from a commercial genomic library as described in Section 2. Restriction mapping (top) was performed with BamHI (B), EcoRI (E), HindII (H), NotI (N), PstI (P), SalI (S) and XbaI (X). Southern blotting with a promoter probe (described in Section 2) was used to identify the 5' regulatory region of this gene that is indicated by the bold arrow (top), marking the direction of transcription of the SERCA2 gene as judged by the presence of exons 4–9. These exons of which the location derived from Southern blotting results is indicated by the bold block. The lower part of the figure denotes the 5' regulatory region that was subsequently sequenced according to the strategy indicated by the thin arrows (plasmids are described in Section 2).

 
For (bi-)directional sequencing purposes, the 5' 1.9 kb BamHI-NotI fragment, the 5' 375 bp BamHI-EcoRI as well as an internal 643 bp EcoRI-EcoRI fragment of the genomic SERCA2 insert were cloned into the respective multiple cloning sites of pBluescript II KS (pBS, Stratagene), resulting in pBS-SPro1.9, pBS-Spro0.4 and pBS-SPro0.6 respectively. For generation of expression vectors harbouring the chloramphenicol acetyltransferase (CAT) gene, the 1.9 kb SalI-NotI fragment of the page insert-DNA was cloned in the SalI site of pCAT-Basic (Promega) after blunting of both fragment and vector with Klenow, resulting in pCAT-Spro1.9. The correct orientation of the promoter with respect to the reporter gene was confirmed by restriction analysis. Subsequently, for sequencing purposes and future expression studies, 5' deletion constructs were generated with Bal-31 nuclease (25 mU/µl) digestion for 10, 20 or 30 min at 30°C of pCAT-Spro1.9 digested with PstI (5' of the promoter). After inactivation of the nuclease, the SERCA2 promoter fragments truncated at the 5' end were cut out of the vector with XbaI, separated on agarose gel, isolated and directionally cloned into EcoRV-XbaI sites of pCAT-basic. From this series of deletion mutants, plasmids with 1.0 and 0.7 kb inserts (pCAT-SPro1.0 and pCAT-SPro0.7) were chosen for sequencing purposes.

Sequencing was performed on an ABI sequencer (Perkin-Elmer), with a dye-terminator cycle-sequencing kit (Perkin-Elmer) and T7 and T3 primers (for pBS constructs) or M13 primers (pCAT-based constructs). Sporadically, dye-primer technology was used as well. For some constructs (especially in sequencing through a GC-rich region surrounding the putative transcription start site), the melting temperature in the PCR cycle was increased from 96 to 99°C, resulting in increased sequence run length (expanded from 200 to 600 bp) and reproducibility. As some deviation was noted with a previously published sequence of a rat promoter fragment [14], all sequence runs were repeated (with a minimum of three runs) until no uncertainty in the 1943 bp nucleotide sequence remained. The sequencing strategy is depicted in Fig. 1 (Bottom).

2.4 Cell culture, DNA transfection and stimulation with agonists
Neonatal rat cardiomyocytes were isolated as described before [15]by eight subsequent trypsinization steps of minced heart tissue. The cells were preplated in complete medium [DMEM/M199 (4:1) with 5% fetal calf serum and 5% horse serum (HS)] in culture flasks (an area of 10 cm2/heart) to increase the purity of the culture. Thereafter, the non-attached cells, mainly cardiomyocytes, were collected, counted and seeded in 20 cm2 culture dishes in complete medium at a density of 7.5x104 cells/cm2. After 24 h, about 90% of the cells were contracting and medium was changed to DMEM/M199 (4:1) supplemented with 5% HS. After 64 h, medium was replaced by 3 ml serum free medium (DMEM/M199; 4:1). Three hours later, ET-1, the {alpha}1-adrenergic agonist phenylephrine (PHE) or AngII was added as indicated in the figures to final concentrations of 10–8, 10–5 and 10–6 M, respectively. These concentrations were shown to elicit maximal signal transduction activity regarding stimulation of phospholipase C in these cells [15, 16].

For promoter activity studies, DNA was lipofected into the cells three hours after switching to serum free medium. To this end, 5 µg of DNA was mixed with 15 µg of the lipofection agent DOTAP (Boehringer Mannheim, Germany) in 20 mM HEPES (pH 7.4) and incubated for 15 min at room temperature before this mixture (75 µl) was added dropwise to the medium. After 18 h, cells were washed, and 3 ml fresh serum free medium was added. After 3 h ET-1 was added to 10–8 M for 48 h to induce hypertrophy.

2.5 Biochemical analysis
After washing the cells with phosphate-buffered saline (PBS), the protein and DNA content of cardiomyocytes were determined in lysates that were prepared by dissolving the cells in 1 N NaOH for 16 h at 4°C. Protein concentration was subsequently determined using the Bradford assay [17], while DNA concentration was measured fluorometrically with 4,6-diamine-2-phenylindol-dihydrochloride [18].

Protein and RNA synthesis were quantified by labelling of cardiomyocytes with either 2 µCi/ml [3H]leucine for 6, 24 or 48 h or with 2 µCi/ml [3H]uridine for 2 h, respectively. Thereafter, the cells were washed with PBS to remove extracellular free label, and cells were fixed and washed three times in 10% TCA for 10 min to remove intracellular unincorporated label. Cells were subsequently lysed in 1 N NaOH for 16 h at 4°C, and incorporated leucine or uridine was quantified by liquid scintillation counting.

Combined transcription–translation regulation of the SERCA2 promoter-CAT reporter plasmid pCAT-Spro1.9 was studied by measurement of the intracellular level of CAT protein. To this end, cells were lysed and CAT protein was quantified by ELISA. All procedures for determination of the CAT protein level were performed according to protocols of the manufacturer (Boehringer Mannheim, Germany).

2.6 RNA isolation and Northern blotting
Total RNA was isolated from the cultured cells according to the guanidine isothiocyanate method (see Unit 4.2 of Ref. [13]). RNA was quantitated by spectrometry at 260 nm, denatured in formamide-loading buffer at 65°C for 5 min and separated on 1% denaturing formaldehyde–agarose gels according to standard protocols. Following electrophoresis and photography, RNA was blotted onto Hybond by upward capillary transfer in 20xSSC. Subsequently, RNA was immobilized by UV-crosslinking (Stratalinker, Stratagene) and rRNA bands were marked.

Probes described below were labelled with [{alpha}-32P]dCTP (3000 Ci/mmol, Amersham) by random hexamer priming (Megaprime kit, Amersham) to a specific activity of about 109 cpm/µg DNA. Northern blots were prehybridized in formamide prehybridization/hybridization solution for 3 h at 42°C. Hereafter, labelled probe was added, and hybridization was continued overnight. Thereafter, the blots were rapidly washed with 2xSSC, 0.1% SDS at room temperature, and subsequently with increasing stringency (0.2xSSC, 0.1% SDS at 42°C and when necessary with 0.1xSSC, 0.1% SDS at 55°C) until background was low. Blots were either exposed to X-ray film (Kodak, X-Omat AR) or exposed in a Molecular Imager (Biorad). The hybridization signal was quantitated either directly in the Molecular Imager or after a densitometric scan (LKB Ultroscan) of multiple exposures on X-ray film to ensure that the signal was in the linear range.

Probes for Northern blotting were generated as follows. A CAT probe (XbaI-NcoI fragment, 662 bp) was excised from pCAT3-basic (Promega). A SERCA2 cDNA (RHCa 117) was obtained from Lompre [19]. Probes for detection of the immediate early genes EGR-1, c-fos, c-jun and c-myc were a kind gift of Dr. H.S. Sharma. Probes for ANF, phospholamban and glyceraldehydephosphate dehydrogenase (GAPDH) (610, 240 and 570 bp, respectively) were generated by RT-PCR on rat heart RNA with primers, containing unique restriction sites, that were designed based on published sequences. These RT-PCR products were cloned in pBluescript, and identity was confirmed by sequencing. Probes were excised from the vector, and isolated from agarose gel before labelling.

To ensure that the hybridization signal with the CAT probe was derived from mRNA and not from contaminating transfected DNA, an RNA sample from transfected cardiomyocytes was treated with DNAse-free RNase, resulting in elimination of the CAT-probe hybridization signal.

2.7 Statistical analysis
Statistical significance was tested by one way analysis of variance, followed by the Student–Newman–Keuls test and was set at {alpha}=0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Isolation of a rat sarcoplasmic reticulum Ca2+ pump gene promoter fragment
As already mentioned in Section 2, Fig. 1 depicts a (partial) restriction map of a genomic fragment containing at least the promoter and exon 4–9 of the rat SERCA2 gene. The promoter-containing 1.9 kb BamHI-NotI fragment was subsequently sequenced according to the strategy depicted at the bottom of Fig. 1. The resulting sequence was aligned to published sequences of the rat [14], rabbit [20]and human [21]SERCA2 promoter fragments using the ClustalW program. As shown in Fig. 2, small deviations were found with the previously reported rat promoter fragment. The high GC content of parts of the 5' regulatory region of this gene prompted us to increase the melting temperature of the cycle-sequencing protocol to obtain 100% certainty in this sequence as described in Section 2. Two regions of high homology (>75%) were found between the rat and rabbit sequences spaced by a 1100 (rat) and 700 bp (rabbit) stretch that displayed low homology. Furthermore, as reported before [21], rat, human and rabbit sequences are nearly identical from –200 to +50.


Figure 2
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  		Fig. 2 Nucleotide sequence of a 1943 bp DNA fragment encompassing the 5' regulatory region and part of exon 1 of the rat SERCA2 gene and homology with previously published sequences. The 5' regulatory region of the rat SERCA2 gene was sequenced as described in Fig. 1. Subsequently, the nucleotide sequence was aligned to previously published sequences of the human [21]and rabbit [20]SERCA2 promoter regions. * Denotes identical nucleotides. Bold, large nucleotides in the rat sequence (five in total) denote sequence differences compared to a previously published rat promoter sequence (–559 to +76; Ref. [14]). The transcription start site, described in the references mentioned above, is labelled +1 and numbering refers to the rat sequence exclusively. The putative CAAT and TATA sequences from the references mentioned above are underlined in the rat sequence. Furthermore, rat, rabbit and human sequences were analyzed individually for putative transcription factor binding sites with the Transfac database using the MatInspector program ([33], internet address http://transfac.gbf.de). Sequences underlined in the bottom sequence depict transcription factor binding sites that are common for the three sequences (or for rat/rabbit where the human sequence is shortest). For further details see the text.

 
To study whether this 1.9 kbp promoter fragment could confer transcriptional regulation normally detected during hypertrophy, a reporter plasmid was constructed harbouring the SERCA2 promoter cloned before the CAT gene (pCAT-Spro1.9). This plasmid was subsequently transfected to cultured neonatal rat ventricular myocytes that have been widely used as a model for agonist- or stretch-induced hypertrophy, and results will be described below.

3.2 Characterization of ET-1 induced hypertrophy of cultured cardiomyocytes
The in vitro hypertrophy model was first characterised by stimulation of cultured ventricular myocytes with either ET-1, PHE or AngII. As can be seen in Fig. 3A, all three agonists induced a significant increase in [3H]leucine incorporation after 24 and 48 h of stimulation. ET-1 showed the strongest response in this respect. This increase in protein synthesis also resulted in increased protein content per cell, and the protein/DNA ratio was significantly increased after stimulation with ET-1 and PHE (Fig. 3B). On the other hand, the slight increase in protein synthesis after stimulation with AngII was reflected a temporal ‘hypertrophic’ effect due to increased protein content per cell after 6 h (not shown). Thereafter, the DNA content was increased, resulting in decreased protein/DNA ratios after 24 and 48 h stimulation with AngII (Fig. 3B). Thus, stimulation with ET-1 proved to be the strongest trigger for induction of hypertrophy in this model (ET-1 versus PHE, 48 h stimulation; P<0.02), as judged by protein content per cell (protein/DNA ratio).


Figure 3
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  		Fig. 3 Increase in protein synthesis (A) and induction of hypertrophy (B) in cultured cardiomyocytes by stimulation with ET-1, PHE and AngII. A. Cardiomyocytes were labelled with [3H]leucine in serum free medium and simultaneously stimulated with either vehicle (control), ET-1 (10–8 M), PHE (10–5) or AngII (10–6 M) for 6, 24 or 48 h. Thereafter, protein synthesis (i.e. leucine incorporation into protein) was quantified as described in the Section 2. Data are expressed as mean±SEM (n=12) relative to unstimulated cardiomyocytes (control). B. Induction of hypertrophy, defined as an increase in protein/DNA ratio, was studied in cardiomyocytes stimulated with the agonists described above. After the indicated period, cells were washed and lysed whereafter protein and DNA contents were determined as described in the Section 2. Values represent mean±SEM versus unstimulated cells for 16 experiments. * Denotes a P value less than 0.05 compared to control, while # denotes a P value less than 0.05 compared to the preceding time point.

 
Not only protein synthesis was increased in these cells, but also total RNA synthesis measured by [3H]uridine pulse labelling, was enhanced (Fig. 4A). Again, stimulation with ET-1 had the strongest effect; already after 6 h RNA synthesis was increased by 58% and after 24 and 48 h an even larger effect was seen. On the other hand, PHE and AngII showed small increases in RNA synthesis after 6 and 24 h, and only after 48 h of stimulation RNA synthesis rate was increased comparably to ET-1. The stimulation of [3H]uridine incorporation was not (exclusively) due to increased turnover rate of RNA, as the RNA content was increased as well (Fig. 4B).


Figure 4
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  		Fig. 4 Increase in RNA synthesis (A) and RNA content (B) in cultured cardiomyocytes after stimulation with ET-1, PHE or AngII. Cultured cardiomyocytes were stimulated simultaneously with either vehicle (control), ET-1 (10–8 M), PHE (10–5 M) or AngII (10–6 M) for 6, 24 or 48 h. For determination of the rate of RNA synthesis, (A), [3H]uridine was added to 2 µCi/ml after this period and incubation was continued for 2 h. Subsequently, cells were washed and lysed and [3H]uridine incorporation in RNA was determined as described in Section 2(data represented as mean±SEM for 12 experiments). For quantification of cellular RNA content, (B), cells were stimulated with agonists as described above. After the indicated periods, cells were washed and RNA was isolated and quantified as described in Section 2. Data are represented as mean±SEM for 4 (6 h stimulation) to 10 (24 and 48 h of stimulation) independent experiments, and are expressed relative to unstimulated control values (23, 24 and 28 µg/dish; 6, 24, and 48 h respectively). * Denotes a P value less than 0.05 compared to control, while # denotes a P value less than 0.05 compared to the preceding time point.

 
Based on the data described above, we subsequently studied transcriptional regulation during hypertrophy of cultured cardiomyocytes induced by ET-1, the strongest agonist in this respect. Fig. 5 shows that directly after stimulation with ET-1, a set of immediate early genes (IEG), coding for well known transcription factors, were transcriptionally activated. The induction of mRNA production was transient, as already after 2 h most IEG mRNA levels had returned to pre-stimulus levels. These early changes were followed at later points in time by alteration of transcription that is reminiscent of in vivo hypertrophy. First of all, in accordance with the increase of total RNA content (Fig. 4B), 48 h of stimulation with ET-1 resulted in increased amounts of mRNA of the house-keeping gene GAPDH as depicted in Fig. 6 [170±18% (n=9) ranging from 120 to 330%, relative to control]. The ANF gene was also transcribed to a higher degree under hypertrophic conditions in these ventricular myocytes; the ANF mRNA level, relative to GAPDH, was increased to 247±48% P<0.02, an increase ranging from 32 to 450%, n=9). Also relative to GAPDH, the levels of phospholamban and SR Ca2+-ATPase mRNA were decreased after 48 h of stimulation with ET-1 (Fig. 6). The level of phospholamban mRNA was decreased to 51±8% (P<0.01, 11 to 89%, n=8, one case of upregulation to 193%) while the SERCA2/GAPDH mRNA ratio declined to 74±7% (P<0.02, 55 to 96%, n=6, one case of upregulation to 130%) after 48 h of hypertrophy induction with ET-1. However, the absolute amounts of phospholamban and SR Ca2+-ATPase mRNA levels, i.e. uncorrected for GAPDH levels, did not change significantly when compared to control.


Figure 5
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  		Fig. 5 Transcription factor expression after stimulation of cultured cardiomyocytes with endothelin-1. Cultured cardiomyocytes were stimulated with ET-1 (10–8 M) for 10, 20, 30, 60 or 120 min. Thereafter, RNA was isolated and Northern blotting was performed as described in Section 2. The resulting hybridization signals for transcription factors EGR-1, c-jun, c-fos and c-myc were normalized to the GAPDH signal to correct for loading and blotting differences. Data represent mean±SEM for 6 experiments and are expressed as percentage of maximal induction for each transcription factor separately. With the exception of EGR-1 at 10 and 120 min, c-jun at 10, 60 and 120 min and of c-myc at 10 min all transcription factor levels were significantly (P value less than 0.05) increased over control (t=O min) levels.

 

Figure 6
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  		Fig. 6 Changes in gene expression during ET-1 induced hypertrophy of cultured cardiomyocytes. Cardiomyocytes, cultured in 20 cm2 dishes in serum free medium, were stimulated with 10–8 M ET-1 for 48 h. Total RNA from each dish was isolated and quantified. Subsequently, 90% of the amount of RNA isolated from each dish was size-fractionated and gene expression was quantified by Northern blotting as described in the Section 2. Hybridization signals are represented relative to unstimulated control cells for GAPDH mRNA, while the ANF, PL and SERCA2 signals were first corrected for increase in total RNA by expressing the ratio relative to GAPDH mRNA before calculating the increase or decrease in expression relative to unstimulated control. Data represent mean±SEM for at least 4 independent experiments. * Denotes a P value less than 0.05 compared to control.

 
3.3 Activity of a SERCA2 promoter fragment during ET-1 induced hypertrophy
Subsequently, we assessed the activity of the SERCA2 gene promoter described in Figs. 1 and 2Go under ET-1 induced hypertrophic conditions as characterized in Figs. 3 and 4Go. Fig. 7 illustrates again that, in a separate series of experiments, ET-1 induced hypertrophy is accompanied by an increase in GAPDH mRNA level (striped bars). This increase was not significantly altered by lipofection of the SERCA2 promoter-reporter plasmid pCAT-Spro1.9 (cross-hatched bars). Moreover, the relative decrease of endogenous SR Ca2+-ATPase mRNA during ET-1 induced hypertrophy was not changed by lipofection of DNA (Fig. 7, compare SERCA2/GAPDH striped versus cross-hatched bars). Transfection of pCAT-Spro1.9 to cultured cardiomyocytes followed by 48 h of hypertrophy induction with 10–8 M ET-1 resulted in a decreased reporter CAT mRNA level (relative to GAPDH) to 56±19% (P<0.05, n=6) when compared to unstimulated transfected cells. Absolute CAT mRNA levels (i.e. uncorrected for GAPDH data) remained unchanged under these hypertrophic conditions when compared to unstimulated cardiomyocytes. Surprisingly, when combined transcriptional and translational activity was assessed by quantification of the CAT protein level, a twofold increase was seen 48 h after the addition of ET-1 resulting in development of hypertrophy (Fig. 7).


Figure 7
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  		Fig. 7 Transcriptional activity of a 1.5 kb-SERCA2 promoter-0.4 kb-exon 1 fragment during ET-1 induced hypertrophy of cultured rat cardiomyocytes. A rat SERCA2 genomic fragment encompassing 1.5 kb of the SERCA2 5' regulatory region and part of exon 1 was isolated and cloned in the reporter vector pCAT-basic as described in Section 2. After overnight transfection of this reporter construct into cultured cardiomyocytes, hypertrophy was induced by stimulation of the cells with 10–8 M of ET-1 for 48 h. Subsequently, gene expression was quantified as described in the legends to Fig. 6 and in Section 2. Hybridization signals are represented relative to unstimulated control cells for GAPDH mRNA, while the (endogenous) SERCA2 and CAT mRNA signals were first corrected for increase in total RNA by expressing the ratio of these mRNAs relative to GAPDH mRNA before calculating the increase or decrease in expression relative to unstimulated control. Furthermore, in a series of independent experiments activity of the SERCA2 promoter was analyzed by quantification of CAT protein with a CAT ELISA as described in Section 2. To asses the influence of transfection on GAPDH and (endogenous) SERCA2 expression, a parallel series was performed to compare expression in untransfected (striped bars) versus transfected cardiomyocytes (cross-hatched bars). Data represent mean±SEM for 4–8 independent experiments. * Denotes a P value less than 0.05.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Cultured neonatal rat cardiomyocytes respond to stimulation by ET-1, PHE and AngII by activating several intracellular signal transduction pathways (reviewed in [22]). As we earlier showed, among these responses are activation of phospholipases C and D [23]resulting in increased intracellular 1,2-diacylglycerol levels [16]and activation of protein kinase C isozymes [23]. Constitutative activation of protein kinase C in cardiomyocytes was shown to result in certain hypertrophic responses like stimulation of the ANF promoter [24], suggesting that the agonists mentioned above can generate signals that lead to induction of hypertrophy.

4.1 Characterization of an in vitro model of hypertrophy
In this study we showed that stimulation of cultured neonatal rat cardiomyocytes with ET-1, {alpha}1-adrenergic agonist PHE or AngII results in an increase in amino acid incorporation after 24 and 48 h. This increased incorporation is due to an increase in protein synthesis and is not solely due to an augmented protein turnover rate, as the protein content per cell (measured as protein/DNA ratio) was increased as well. In the presence of ET-1 or PHE, DNA levels per culture dish did not change (data not shown) and hypertrophy developed progressively after stimulation with ET-1. It was surprising to note, however, that after 6 h stimulation with AngII, when protein synthesis was not significantly increased, the protein/DNA ratio had already increased by 15%, suggesting that the protein turnover rate was modified by this agonist as well. After 24 and 48 h of stimulation with AngII, the protein/DNA ratio was not any longer significantly increased despite increased protein levels, which was due to increased DNA content per culture dish (data not shown). It has indeed been reported that AngII can have several (opposing) effects on cells, brought about by the presence of two different receptor subtypes (AT1 and AT2) that are coupled to growth- and antigrowth responses, respectively [25]. Therefore, induction of hypertrophy by AngII cannot be judged with certainty from protein and DNA data in this respect.

As was shown before, hypertrophy of cultured cardiomyocytes is not only accompanied by increased cell size and development of a well-ordered contractile apparatus, but also by increased transcriptional activity in in vivo [26]as well as in in vitro [27]models leading to increased total RNA levels. We could show that after 6 h, when protein synthesis was not yet significantly increased, incorporation of [3H]uridine into RNA was already increased by 58 and 21% after stimulation with ET-1 and PHE, respectively. After 24 h of stimulation with ET-1, uridine incorporation had reached a maximal increase of 100%. PHE and AngII-induced effects on uridine incorporation were slower in onset, with only 26 and 13% increase after 24 h. However, prolonged incubation with these agonists also resulted in a large increase in transcriptional activity (85 an 92% increase in uridine incorporation). Because, in contrast to protein, the halflife of mRNA is relatively short (t1/2 about 30 min), increased uridine incorporation in RNA during pulse labelling for 2 h might represent either augmented RNA synthesis or increased turnover. Although, in contrast to protein and DNA, RNA levels were highly variable between experiments (probably due to the multi-step isolation protocol, including precipitation), we could show that stimulation with ET-1 and PHE led to increased transcriptional activity resulting in increased RNA levels per cell after 24 (and 48) h. As the large (58%) increase in uridine incorporation after 6 h of stimulation with ET-1 was not reflected in increased RNA levels, it seems likely that not only RNA synthesis but also RNA turnover rate is influenced by ET-1. Increases in RNA level after stimulation with AngII were too low and variable to reach significance.

These biochemical data indicate that ET-1 is a strong inducer of hypertrophy in cultured rat cardiomyocytes. This was also reflected in our study in changes in transcription that are associated with hypertrophy. As was shown before [28]stimulation of rat cardiomyocytes with ET-1 leads to induction of transcription of several so-called immediate early genes (IEG) that code for transcription factors. Already after 10 min, c-jun mRNA levels were increased, followed at 20 min by EGR-1 and c-fos. These three transcription factors were maximally but transiently induced at 30 min, and had returned to base level approximately 2 h after addition of ET-1. Not all transcription factors were induced in the same time scale as c-fos, EGR-1 and c-jun; induction of c-myc was slower in onset, reached a maximum at 60 min and had decreased to about 50% of maximal induction at 120 min.

Although not all details are known yet with respect to transcription regulation by IEG genes, it was established that c-fos and c-jun regulate the activity of the promoter of the ANF gene [29, 30]. This ANF gene, normally only expressed in the ventricle in the fetal state, is induced during development of hypertrophy in several in vitro and in vivo models. Also in our model of hypertrophy, stimulation with ET-1 for 48 h led to increased ANF levels but the effect was variable. First of all, we chose to relate the level of ANF mRNA to that of the ‘housekeeping’ gene GAPDH. The level of this GAPDH mRNA was increased after 48 h of stimulation with ET-1 to 170%, in accordance with the general increase of 60% in RNA content after this period. Secondly, although ANF expression in the ventricle in vivo is (almost) absent, unstimulated cultured ventricular cardiomyocytes express ANF to a low, but variable level.

Development of hypertrophy and failure is associated with several phenotypic changes, including the above described induction of ANF. Furthermore, expression of the phospholamban (PL) gene and the activity of the SR Ca2+ pump are depressed [5]. The cause of the latter phenomenon is still controversial. SERCA2 mRNA levels are decreased in hypertrophy/failure and several studies report decreased Ca2+ pump protein levels as well, but other reports do not confirm this latter finding [2–4]. It was therefore important to characterize the PL and SERCA2 mRNA levels in our model as well. We found a decrease in PL as well as SERCA2 mRNA level when compared to GAPDH after 48 h of stimulation with ET-1. However, as GAPDH mRNA is increased concomitant with a general increase in RNA level, this ‘downregulation’ of PL and SERCA2 is not an absolute one. When expressed on a cellular level [i.e. per culture dish where cell number (DNA content) is not increased by stimulation with ET-1], both mRNA levels remain relatively constant during development of hypertrophy. Thus this depicts a situation where some genes are specifically induced (e.g. IEG and ANF) in addition to a general increase in transcriptional activity (total RNA and GAPDH) during hypertrophy. On the other hand, some genes like e.g. PL and SERCA2 do not respond to this general increase in transcriptional activity during hypertrophy, resulting in a relative downregulation.

Despite the fact that SERCA2 mRNA showed only a relative down regulation, we showed in the past that SR Ca2+ pump activity (expressed per mg cellular protein) is decreased under these exact conditions (i.e. ET-1 induced hypertrophy of cultured cardiomyocytes), independent of regulation of Ca2+ pump activity by PL [15]. Therefore it is reasonable to assume that SERCA2 mRNA and protein levels are similarly regulated in this model.

4.2 SERCA2 promoter activity in hypertrophic cardiomyocytes
The isolated rat promoter for SERCA2 (–1580 to +363) cloned before a reporter gene and transfected to cardiac myocytes, responded to ET-1 induced hypertrophy in a manner similar to the endogenous SERCA2 gene. Levels of reporter CAT mRNA were decreased after 48 h of hypertrophy induction with ET-1. However, this decrease was only observed when compared to the (increased) GAPDH mRNA level. This suggests that this 1943 bp SERCA2 DNA fragment contains all relevant genetic information that is normally responsible for the relative downregulation of SERCA2 gene expression during ET-1 induced hypertrophy of cultured rat cardiomyocytes. In contrast to the decreased CAT mRNA levels, CAT-protein (with a half life of about 40 h) was increased after 48 h of stimulation with ET-1. In this case, either CAT-mRNA (and thus SERCA2 promoter activity) was increased above control early in the hypertrophic phase, or the CAT mRNA (with only a short part of the SERCA2 mRNA 5' UTR) is efficiently transcribed under hypertrophic conditions. No data are yet available to distinguish between these transcriptional or translational events.

The isolated 1943 bp rat promoter showed a nearly perfect homology with a previously published part of the rat promoter sequence (–559 to +76) [14]. Only 5 base deviations were detected, that furthermore were often in accordance with published rabbit and human promoter sequences [20, 21]. A previously unidentified 210 bp region of high homology was detected with the rabbit SERCA2 promoter region. Thus, by alignment, only two regions with significant homology (arbitrarily set at >75%) were found: nt –1520 to –1310 of the rat sequence with rabbit and nt –220 to +363 of the rat sequence with human and rabbit promoters. In the region of homology between the rat and rabbit sequences an additional TATA-box (TAATTAAA, around –1416) and CAAT box (around –1460) were detected. However, a sequence search in Genbank did not reveal any homology with known genes. Furthermore, the low homology directly downstream of the TATA box suggests that these sites are not directing transcription from a common gene. Analysis of the rabbit, rat and human SERCA2 promoter regions for transcription factor bindings sites as described in the legends to Fig. 2 revealed only a limited number of common cis-acting elements, namely GATA (–1465 bp) and Nkx2.5 (–1420) for rabbit/rat and CDPR3HD (–206), GC-rich regions (SP1 sites) (–200, –183 and –119), a CAAT site (–83), an USF binding site (–64) and a GATA site (–29 bp) for human/rat/rabbit (all underlined in Fig. 2).

Although the transcription start site of the SERCA2 gene was not identified experimentally in this study, it was reported in human and rat studies [14, 21]that besides the start site labelled +1 in our study, a second mRNA may arise from a position (9–10 nt) downstream of the +1 site. Indeed, analysis of the promoter sequence with a promoter prediction neural network program (Ref. [31], internet address http://www-hgc.lbl.gov/projects/promoter.html) indicated that a transcription start site was present at 8 nt downstream of the site labelled +1. Computer analysis of the relative strength of these promoters based on homology with the perfect TATAAA sequence and binding and activity data [32]showed that these sites both have a relatively low predicted transcriptional activity of 0.468 (+1 transcription start site, GATAAAT sequence at –25) and 0.305 (+9 start site, TATTAGA at –16). Further analysis of the sequences indicated that the phospholamban promoter, CATAAGA, also had a low predicted transcriptional strength (0.292), when compared with the ANF and skeletal {alpha}-actin promoters which have an optimal TATAAAA box (predicted strength of 1.000). As not many common transcription factor binding sites are present in these three SERCA2 promoters [33]that might be responsible for the observed relative downregulation of the expression of the SERCA2 gene, one might hypothesize that the TATA box strength is an important factor in determining the mRNA level during the general increase in transcriptional activity in a hypertrophic state. However, this hypothesis remains to be tested by mutational analysis of both TATA boxes present in the SERCA2 promoter. Furthermore, we are now testing truncated SERCA2 promoter fragments for transcription activity during ET-1 induced hypertrophy of cultured cardiac myocytes. To this end we are employing pCAT-SPro1.0 and pCATSPro0.7 (see Section 2) that contain 646 and 305 bp respectively of the 5' regulatory region of the SERCA2 gene coupled to 363 bp of the 5' UTR of the SERCA2 mRNA. Together these experiments will have to learn whether either cis-acting elements or the TATA box are responsible for the relative downregulation of the transcription of the SERCA2 gene during (ET-1 induced) hypertrophy.

Time for primary review 27 days.


   Acknowledgements
 
This study was supported by the Netherlands Heart Foundation grants M93.004 and 95.109 (Drs. H.A.A. van Heugten and K. Eizema) and D94.001 (M.C. van Setten).


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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