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Cardiovascular Research 2004 63(3):537-544; doi:10.1016/j.cardiores.2004.04.005
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Copyright © 2004, European Society of Cardiology

Contractile arrest reveals calcium-dependent stimulation of SERCA2a mRNA expression in cultured ventricular cardiomyocytes

Ronald Vlasblom, Alice Muller, René J.P Musters, Marian J Zuidwijk, Cornelis van Hardeveld, Walter J Paulus and Warner S Simonides*

Laboratory for Physiology, Institute for Cardiovascular Research (ICaR-VU), VU University Medical Center, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands

* Corresponding author. Tel.: +31-20-4448116; fax: +31-20-4448255. Email address: ws.simonides{at}vumc.nl

Received 30 December 2003; revised 19 March 2004; accepted 7 April 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Objective: Downregulation of sarco-endoplasmic reticulum calcium ATPase 2a (SERCA2a) expression is a critical marker of pathological myocardial hypertrophy. The effects of calcium-dependent signaling and of contractile activity on the regulation of myocardial SERCA2a expression remain unclear. The present study dissociates effects of calcium-dependent signaling through calcineurin (CN) and calmodulin dependent protein kinase-II (CAMK-II), from effects of contractile activity in spontaneously contracting rat neonatal ventricular cardiomyocytes (NVCM) using 2,3-butanedione monoxime (BDM), which arrests contractions but maintains calcium fluxes. Methods: SERCA2a mRNA expression was analysed using Northern hybridisation in spontaneously contracting NVCM (control) and in NVCM treated with either BDM, L-type Ca2+-channel blocker (verapamil), CN-blocker (cyclosporin A; CsA), CAMK-II blocker (KN-93), or combinations thereof. Transient transfection of the CN-dependent transcription factor nuclear factor of activated T-lymphocytes (NFATc), coupled to GFP, was used to detect NFAT nuclear translocation. The effects of CN/CAMK-II-dependent signaling were further dissected into effects of the transcription factors NFATc4 and myocyte enhancer factor 2c (MEF2c) on the activity of various SERCA2a promoter fragments using transient transfection assays. Results: Treatment with BDM induced a 2.5-fold rise in SERCA2a mRNA, which was abolished by addition of verapamil and was reduced by addition of CsA (–40%) and KN-93 (–20%). NFAT nuclear translocation was similar in control and BDM-treated NVCM. SERCA2a promoter activity was stimulated by NFATc4 and MEF2c, but only when both factors were co-transfected. Conclusion: Following contractile arrest with BDM, upregulation of SERCA2a mRNA expression by CN/CAMK-II signaling becomes evident. This upregulation is likely the result of synergistic stimulation of SERCA2a promoter activity by NFATc4 and MEF2c. Contractile activity opposes this upregulation through distinct and independent pathways.

KEYWORDS SERCA2a; Gene expression; Cardiac hypertrophy; Calcium; Signal transduction; Contractile activity


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Cardiac hypertrophy occurs in response to a chronic increase in workload and is aimed at normalizing wall-stress. When unsuccessful, the initially adaptive hypertrophy will evolve to a pathological form characterized by changes in cardiac gene expression including downregulation of the sarco-endoplasmic reticulum calcium ATPase 2a (SERCA2a) protein level [1,2]. This downregulation may affect calcium handling and contribute to contractile dysfunction, as suggested by the improved contractility of hypertrophied myocardium following SERCA2a protein overexpression using transgenic approaches [3,4]. The reduction of myocardial SERCA2a mRNA and protein expression in pathological hypertrophy was attributed to reduced SERCA2a promoter activity [5]. This finding was further corroborated by lower activity of a –1800 bp SERCA2a promoter fragment when transfected in vivo in pressure-overloaded, hypertrophic hearts [6,7].

Possible mediators of reduced SERCA2a expression in pressure overload-induced myocardial hypertrophy include calcium-dependent signaling pathways involving calcineurin (CN) and calmodulin-dependent protein kinase (CAMK) [8], as wells as calcium-independent signaling pathways directly linked to the increased mechanical stress [9]. Stimulation of the calcium-dependent signaling pathways is considered to be important in the development of myocardial hypertrophy because of the demonstration of increased activity of CN and CAMK during pressure overload [10,11]; because of the development of massive cardiac hypertrophy following cardiac-specific overexpression of CN or CAMK [12,13]; and because of the ability of CN or CAMK blockers to inhibit development of hypertrophy both in vitro and in vivo [12,14,15].

The CN and CAMK pathways modulate transcription through activation of the transcription factors NFAT (nuclear factor of activated T-lymphocytes) and MEF2 (myocyte enhancer factor 2), respectively. However, the effects of CN or CAMK signaling on SERCA2a expression remain unclear. In myocardial hypertrophy induced by CN overexpression, both a fall in SERCA2a mRNA [12] and a rise in SERCA2a protein [16] have been observed. Moreover, control of SERCA2a promoter activity by CN or CAMK signaling has so far not been explored. It is similarly unclear what the role of mechanical activity is in the regulation of SERCA2a transcription.

Here we investigated the effects of calcium-dependent signaling in the presence or absence of contractile activity on myocardial SERCA2a mRNA expression. Because of the obligatory linkage in the in vivo setting of calcium fluxes and contractile activity, their individual effects can only be explored in the in vitro setting. The present study therefore used spontaneously contracting neonatal ventricular cardiomyocytes (NVCM). Effects of calcium-dependent signaling were studied either following treatment of cells with 2,3-butanedione monoxime (BDM), which maintains calcium fluxes but abolishes contraction, or following treatment with verapamil, which inhibits both calcium fluxes and contraction. Furthermore, transient transfection assays of NVCM were used to directly investigate transcriptional regulation of the SERCA2a promoter by NFATc4 and MEF2c.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusions
 References
 
2.1. Cell culture of rat neonatal ventricular cardiomyocytes (NVCM)
All animals were treated in accordance with the national guidelines of the Institutional Animal Care and Use Committee of the VU University medical center. These guidelines conform with the Guide for the Care and Use of Laboratory animals published by the US National Institutes of Health (NIH Publication No.85-23, revised 1996). Neonatal ventricular myocytes were isolated from 2- or 3-day-old Wistar rats (Harlan), essentially as described by Iwaki et al. [17]. Pups were decapitated after CO2 anaesthesia and hearts were quickly removed. The ventricles were digested by collagenase Type II (Worthington Biochemicals) and pancreatin. Ventricular myocytes were plated on 1% gelatine-coated dishes at a confluent density of 7 x 104 cells/cm2 (day 0). Cells were plated for 2 days in DMEM containing 10% horse serum and 5% foetal bovine serum. At day 3 serum was replaced by DMEM containing 0.2% BSA. Starting at day 4, medium was changed and supplemented with blockers. Cells were harvested at day 7. Medium, including blockers, was changed once at day 5. Cells were allowed to contract spontaneously, were contraction arrested using verapamil (10µM) (Sigma), or were contraction arrested, while maintaining calcium fluxes using the cross-bridge uncoupler BDM (7.5mM) (Sigma). The role of CN and CAMK-II in SERCA2a mRNA expression was investigated using the inhibitors cyclosporine A (CsA; 1µM) (Calbiochem) and KN-93 (2 µM), respectively (Sigma). Cells were kept in a humidified chamber with 5% CO2 at 37 °C. 1-[β-D-arabinofuranosyl]cytosine (10 µM) was present from day 3 till day 5; 1% penicillin/streptomycin were present in the media during the entire culture period.

2.2. RNA isolation and northern hybridisation
Isolated total RNA (Tripure, Boehringer Mannheim) was separated on a 1.2% formaldehyde agarose gel (10 µg each sample; 40 V, 4,5 h). RNA was transferred by capillary flow onto Hybond-N-Membrane (Amersham), and cross-linked with ultraviolet radiation. Blots were pre-hybridised for 1 h in 5 x SSC (0.8 M NaCl, 0.08M C6H5Na3O7·2 H2O), 5 x Denhardt's (100 x Denhardt's is 2% ficoll, 2% polyvinylpirrolidone, 2% BSA) 60 µg/ml sheared herring sperm DNA, 1% SDS, 50% formamide, after that a specific rat SERCA2a cDNA probe (1kb[PstI (+2624) to (polyA)+-tail]) was added. The probe was random prime labelled with [{alpha}-32P] dCTP (3000 Ci/mmol) (Amersham) using the High Prime labelling kit (Roche). Blots were hybridised for approximately 16 h and then washed to a stringency of 0.1 x SSC+0.1% SDS at 42 °C. Analysis was performed by phosphoimaging and messengers were quantified by the computer program Image Quant (Bio-Rad).

2.3. Preparation of plasmids for transfection
Three SERCA2a promoter fragments with 5'-ends at –6588, –3263, –550 bp and 3'-ends at +550 (+1 is the transcription initiation site) were cloned into a promoterless pGL3-basic luciferase vector (Promega, Madison, USA). CMV-pRL (Renilla) (Promega) was used for normalisation. Plasmids expressing constitutively active NFATc4, NFATc-GFP and wild type MEF2c were generous gifts from Dr. E.N. Olson [18].

2.4. Sequence analysis of the rat SERCA 2a promoter
The rat SERCA 2a promoter sequence was subcloned from a rat genomic P1 clone obtained from Genome Systems (St. Louis, USA). The 5'-sequence of the SERCA2a gene (6588 bp) was sequenced on both strands by BaseClear (Leiden, The Netherlands). Using the Blast search program (http://www.ncbi.nlm.nih.gov/genome/seq/RnBlast.html), the promoter sequence was compared to the rat genome sequence and identified as the SERCA2a promoter. Furthermore, the 6588 bp SERCA2a fragment of the rat was compared to the SERCA2a promoter sequence of the mouse. Conserved regions in both promoters were present up to –4100 bp upstream. Putative binding sites for the NFAT and MEF transcription factors were localized using the internet program CONSITE and TRANSFAC (http://mordor.cgb.ki.se/cgi-bin/CONSITE/consite?rm=t_input_single and http://transfac.mirror.edu.cn/TRANSFAC).

2.5. Transient transfection assays
Neonatal ventricular myocytes were transfected with Fugene (Roche). SERCA2a luciferase promoter constructs (250 ng) were transfected together with CMV-pRL plasmids (25 ng), with or without plasmids expressing constitutively active NFATc4 (1 µg), MEF2c (1 µg). Empty pOCAT plasmid was added to the transfection mix to equalise total DNA during transfection. Two days after transfection, cells were washed with phosphate buffered saline (PBS), harvested in 200 µl Luciferase lysis buffer (Promega). Suspensions were spun down for 2 min at 10,000 x g and the supernatant was used directly for analysis. Promoter activities were determined using the Dual Luciferase assay kit (Promega). Light signals were detected with a luminometer (Berthold).

For live-cell imaging experiments, spontaneously contracting NVCM were transfected with 1.5 µg NFATc-GFP. Twenty-four hours after transfection cells were either kept in medium containing 0.2% BSA and contracted spontaneously, or were arrested using either BDM (7.5 mM; either with or without 1µM CsA), or verapamil (10 µM). Twenty-four hours after the addition of the blockers, the cells were analyzed for NFATc-GFP nuclear translocation. Nuclear localization of NFAT-GFP was confirmed by co-localization with Hoechst 33342 staining. Nuclear NFAT-GFP was quantified by measuring the sum of intensities, i.e., mean intensity of fluorescence times area in pixels, using Slidebook TM software as described under 2.6.

2.6. Digital imaging microscopy
All live-cell experiments were performed on a ZEISS Axiovert 200 MarianasTM inverted microscope (Intelligent Imaging Innovations, Denver), equipped with a motorized stage (stepper-motor z-axis increments: 0.1 µm), and a turret of four epi-fluorescence cubes (including DAPI, narrow-band GFP [Chroma Technology, Rockingham, VT] as well as a brightfield DIC cube). A cooled CCD camera (Cooke Sensicam [Cooke, Tonawanda, NY], 1280 x 1024 pixels) recorded images with true 16-bit capability. The camera is linear over its full dynamic range (up to intensities of over 4000) while dark/background currents (estimated by the intensity outside the cells) are typically <100. Exposures, objective, montage and pixel binning were automatically recorded with each image stored in memory (Dell Dimension workstation: 1.7 GHz Xenon® dual processor, 2 GB RAM). The microscope, camera and all other hardware settings were controlled by SlidebookTM software (Slidebook version 4.0 [Intelligent Imaging Innovations]). Microscopy was performed with a custom 10 x air lens (ZEISS). The motorized filter turret allowed acquisition of one composite image on darkfield and brightfield within 2 s.

2.7. Statistical analysis
Data were expressed as means and evaluated using ANOVA with Bonferoni as a post-hoc analysis. Differences were considered significant at p<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusions
 References
 
3.1. Effects of calcium fluxes and contractile activity on SERCA2a mRNA expression
Total RNA was isolated from control (CTR) NVCM, which contracted spontaneously, from NVCM treated with BDM, which showed no contractile activity, from NVCM treated with verapamil, which also showed no contractile activity and from NVCM treated with either a CAMK-II inhibitor (KN-93) or with the CN-inhibitor CsA, which both maintained contractile activity. Only BDM treatment resulted in a significant 2.5-fold increase in SERCA2a mRNA expression (Fig. 1A). KN-93 as well as CsA treatment induced a modest, non-significant decrease in SERCA2a mRNA expression (Fig. 1A). In line with the load-induced expression of atrial natriuretic factor (ANF), both in vivo and in cultured NVCM [8,19], contractile arrest with either BDM or verapamil resulted in a 70% decrease in ANF mRNA level (data not shown).


Figure 1
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  		Fig. 1 (A) Upper panel: representative autoradiograph of a SERCA2a and GAPDH Northern hybridization showing the effects of calcium signaling and contractile activity on SERCA2a mRNA expression. Total RNA (10 µg/lane) was size fractionated and hybridised with {alpha}32P-dCTP labelled probes for SERCA2a GAPDH mRNA. Lower panel: arrest of contractile activity, but maintenance of calcium fluxes using (BDM) causes a 2.5-fold increase in SERCA2a mRNA expression, as compared to the spontaneously contracting NVCM (CTR). Treatment of NVCM with verapamil, CsA or KN-93 did not result in a change in SERCA2a mRNA expression. All blockers or combinations of blockers were added at 4 days after isolation and were present during 3 days. Data are presented as means ±SEM of five independent experiments. {dagger}: p<0.001 vs. CTR. Abbreviations: CTR=control (spontaneous contractions), BDM=2,3-butanedione monoxime, vera=verapamil, CsA=cyclosporine A. (B) Upper panel: representative autoradiograph of a SERCA2a and GAPDH Northern hybridization showing the involvement of calcineurin and CAMK-II in the 2.5-fold stimulation of SERCA2a mRNA expression. Lower panel: The 2.5-fold increase in SERCA2a mRNA expression, as observed in the mechanically unloaded cells (BDM), was abolished after addition of verapamil and could be partly (20%) inhibited by the addition of KN-93, a CAMK-II inhibitor. Furthermore, addition of CsA to the BDM-treated cultures resulted in a 40% inhibition. All blockers or combinations of blockers were added at 4 days after isolation and were present during 3 days. Data are presented as means ± SEM of five independent experiments. {dagger}*p<0.001 vs. CTR, {dagger}p<0.001 vs. BDM; {ddagger}p<0.05 vs. BDM, *p<0.001 vs. BDM.

 
Addition of verapamil to BDM-treated cells abolished the BDM-induced increase in SERCA2a mRNA (Fig. 1B). The BDM-induced increase in SERCA2a mRNA was reduced by 20% following addition of KN-93 and by 40% following addition of CsA (Fig. 1B), suggesting a CN/CAMK-II-dependent stimulation of SERCA2a expression in BDM-arrested NVCM.

3.2. NFATc4/MEF2c signaling and SERCA2a promoter activity
Various lengths of the rat SERCA2a promoter (–550, –3263 and –6588 bp) driving expression of a luciferase reporter gene were transfected either alone or in combination with plasmids expressing the transcription factors NFATc4 and/or MEF2c. The transcription activities of the different SERCA2a promoter lengths were unchanged after co-transfection of either NFATc4 or MEF2c alone (Fig. 2B–D). Co-transfection of both NFATc4 and MEF2c resulted in a synergistic repression of the –550 and –3263 bp promoter fragments (Fig. 2B,C) and a synergistic stimulation of the –6588 bp promoter fragment (Fig. 2D). This synergistic stimulation supports the aforementioned CN/CAMK-dependent stimulation of SERCA2a mRNA expression.


Figure 2
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  		Fig. 2 Panel A: schematic linear representation of the various lengths of the SERCA2a promoter fragments that were cloned in the pGL3 luciferase reporter plasmid. Sequence analysis of the promoter revealed several NFAT concensus sites (N), and three MEF consensus sites (M). The M site indicated in bold, was fully conserved in both rat and mouse SERCA2a promoter. The complete rat promoter sequence was aligned both with the known rat as well as the mouse SERCA2a promoter sequence and showed high homology up to 4100 bp upstream (hatched boxes indicate >85% sequence homology). Panels B–D: effects of NFATc4 and MEF2c on SERCA2a promoter activity in NVCM cultures. Plasmids containing 0.55 kb (B), 3.3 kb (C) and 6.6 kb (D) of the SERCA 2a promoter sequence, driving the luciferase gene, were co-transfected with expression plasmids for either NFATc4 or MEF2c, or in combination. Luciferase activities were normalized to Renilla activities. The average basic activity of the three SERCA2a promoter fragments decreased with increasing length of the promoter sequence. The activity of the 6.6 kb fragment is approximately 60% less active than the 0.55 kb fragment. Data are presented as means±SEM of six independent experiments. {dagger}p<0.01 vs. basal promoter activity; {ddagger}p<0.01 vs. NFAT co-transfection, *p<0.01 vs. MEF co-transfection.

 
NFAT consensus sites were identified in all three SERCA2a promoter lengths, but MEF consensus sites were only identified in the –6588 bp promoter fragment. This finding could underlie the synergistic stimulation by NFATc4 and MEF2c of the –6588 bp promoter fragment. To discern conserved regions of the SERCA2a promoter, the –6588 bp sequence was aligned with the same length of the 5'-SERCA2a promoter sequence of the mouse. This revealed extensive sequence homology up to –4100 bp (hatched boxes in Fig. 2A). Notably, one MEF consensus site at position –3713 was fully conserved (Fig. 2A). Conservation of this MEF site underscores its importance for the synergistic stimulation by NFATc4 and MEF2c of the SERCA2a promoter.

3.3. NFAT translocation in NVCM cultures
Spontaneously contracting NVCM were transfected with a NFATc-GFP expression plasmid to analyse nuclear translocation of NFAT under various conditions. Using digital imaging microscopy, the sum intensity, of nuclear GFP signals in CTR NVCM and in BDM-treated NVCM were similar. There was, therefore, no evidence for contraction-induced inhibition of nuclear translocation of NFAT. Both verapamil and CsA treatments reduced nuclear translocation of NFAT, consistent with the calcium-calcineurin-NFAT signaling cascade (Fig. 3).


Figure 3
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  		Fig. 3 Effect of contractile activity and calcium-dependent signaling on nuclear translocation of NFATc-GFP in NVCM. Upper panel: representative life cell images of NVCM cultures transfected with an NFATc-GFP-expression plasmid. BDM, CsA and verapamil were added 24 h after transfection and cells were analyzed 24 h later. Panels A–D show bright-field images with superimposed NFATc-GFP signal; panels E–G show NFAT-GFP signals only. Lower panel: quantification of nuclear NFATc-GFP. The sum intensities of nuclear GFP-signal of eight different cells from three independent experiments are shown. Data are presented as means±SEM. {dagger}p<0.05 vs. BDM; {ddagger}p<0.05 vs. CTR.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusions
 References
 
The present study on spontaneously contracting NVCM observes a CN/CAMK-II-dependent upregulation of SERCA2a mRNA expression which becomes evident following contractile arrest. Transient transfections of various lengths of the rat SERCA2a promoter suggest that the CN/CAMK-II-induced upregulation of SERCA2a mRNA expression results from synergistic stimulation of the SERCA2a promoter by NFATc4 and MEF2c. Life cell digital imaging microscopy shows similar NFAT nuclear translocation in spontaneously contracting and BDM-arrested cardiomyocytes, but lower NFAT nuclear translocation in verapamil-arrested or CsA-treated cardiomyocytes. This suggests SERCA2a mRNA expression to be controlled by calcium and contractile activity through distinct and independent pathways, at least with respect to CN/NFAT signaling.

4.1. Upregulation of SERCA2a mRNA expression by contractile arrest
Numerous studies revealed myocardial mechanical overload to reduce SERCA2a promoter activity and mRNA or protein expression [1,5–7,20] and this reduction of SERCA2a expression has become a marker for development of pathological hypertrophy during chronic mechanical overload. The spontaneously contracting NVCM used in the present study probably operated at an elevated mechanical load as they resisted deformation resulting from anchoring to the gelatine-coated dish and from simultaneously contracting neighbouring cells in the confluent culture, as also noted by others using this model [20,21]. The increase in SERCA2a mRNA expression following contractile arrest of these cells with BDM is in line with the in vivo and in vitro analyses describing load-dependent expression of SERCA2a [1,6,7,20,21]. Mechanical overload-induced repression of SERCA2a promoter activity has also been observed following in vivo myocardial transfection of a –1800 bp SERCA2a promoter fragment, which revealed significantly lower promoter activity in pressure-overload hypertrophy than in control hearts [6,7]. The reverse, namely restored SERCA2a protein expression following mechanical unloading has recently been reported in end-stage human heart failure after insertion of a left ventricular assist device (LVAD) [22]. The inhibitory effect of mechanical load on SERCA2a promoter activity may result from the reduced contribution of load-dependent factors binding to cis-acting sites in the SERCA2a promoter, such as GATA4, NFkB and SRF, which are stimulated by PI3-kinase, reactive oxygen species and RhoA, respectively [23–25]. Our data indicate that the putative load-dependent pathways do not interfere with the nuclear translocation of NFAT, as part of the CN/NFAT-dependent stimulation of SERCA2a expression (see below), since nuclear levels of NFAT were the same in contracting and BDM-arrested cells. However, interference of load-dependent pathways with events down-stream of activated CAMK cannot be excluded based on our data.

4.2. Calcium-dependent stimulation of SERCA2a mRNA expression in BDM-arrested NVCM
The role of calcium-dependent signaling in the regulation of myocardial SERCA2a expression is controversial. In CN overexpression-induced myocardial hypertrophy both a fall in SERCA2a mRNA [12] and a rise in SERCA2a protein were observed [16]. Furthermore, control of SERCA2a promoter activity by CN/CAMK-II signaling has not been explored. The present study showed significant reductions in the elevated SERCA2a mRNA content of BDM-arrested NVCM following administration of a CAMK-II inhibitor (KN-93) or of a CN inhibitor (CsA). In control NVCM, there was only a trend toward lower SERCA2a mRNA levels following administration of KN-93 or of CsA. This probably resulted from the contraction-induced lowering of the SERCA2a level in the control NVCM, which rendered additional inhibitory effects less detectable. Nevertheless, the current findings indicate calcium-dependent signaling to result in upregulation of SERCA2a mRNA expression. Although a rise in SERCA2a mRNA expression is not necessarily related to a concomitant rise in protein expression [26], our data are consistent with the previously reported rise in SERCA2a protein in CN overexpression-induced myocardial hypertrophy [16], and also with the fall in SERCA2a protein observed in CsA-treated skeletal muscle [27]. Moreover, the present observations are the first to document CAMK-II-induced upregulation of SERCA2a mRNA expression. This extends the role of CAMK-II signaling in myocardial hypertrophy [11,13,28] beyond its previously reported actions on SERCA2a and phospholamban phosphorylation [29–31] to regulation of expression of SERCA2a.

The stimulatory effect of calcium-dependent signaling on SERCA2a mRNA expression was also analysed at SERCA2a promoter level using transient transfection assays. Various cis-acting sites have been described to be involved in stimulation (thyroid-hormone response elements [32], MCAT, CarG, E-box [33]) or inhibition (endoplasmic stress response elements [34], Sp-1 [35], Egr-1 [36]) of SERCA2a promoter activity. However, the present study focussed on the analyses of the effects of NFATc4 and MEF2c on various lengths (–550, –3263 and –6588 bp) of the SERCA2a promoter. Effects were only observed when both factors were present, showing inhibition of activity of both shorter fragments, which appears to be in agreement with the inhibitory effects of hypertrophic stimuli on the –1800 bp fragment of the SERCA2a promoter in vivo [6,7]. The mechanism for suppressed activity of the shorter fragments by a combination of both factors but not by a single factor remains unresolved. Unexpectedly, a synergistic 3-fold stimulation in activity of the longest promoter fragment (–6588 bp) was observed. Similar synergistic or additive actions of NFAT and MEF have also been described for transcription of other genes [18,37]. The stimulation of the –6588 bp SERCA2a promoter by the combination of NFATc4 and MEF2c supports the observed calcium-dependent upregulation of SERCA2a mRNA expression. The stimulatory effect on the long fragment, but not on the –3263 bp fragment, is most likely related to the presence of consensus binding sequences for one or both factors in the sequence upstream of position –3263. Sequence analysis identified NFAT and MEF consensus sites at approximately –3500 bp (see Fig. 2A). The importance of this region is supported by analysis of sequence homology of the rat and mouse SERCA2a 5'-sequences, which showed high homology up to –4100 bp, but not beyond. In particular, the MEF-site was fully conserved (see Fig. 2A; indicated with M, written in bold). All previous studies addressing SERCA2a promoter activity with respect to hypertrophic signaling used promoter fragments of maximally 3200 bp in length [6,7,38]. Results from these studies may therefore not show the full effect on SERCA2a promoter activity, given the opposite response to CN/CAMK-signaling of such fragments compared to the longer promoter sequence (Fig. 2A–D).

4.3. Study limitations
This in vitro study describes the stimulation of SERCA2a mRNA expression upon contractile arrest, with a potential role for NFATc4 and MEF2c as synergistic activators of SERCA2a transcription. However, the physiological role of this calcium-dependent stimulation of SERCA2a expression and the involvement of NFATc4 and MEF2c, especially in the context of an increased in vivo mechanical load, remains to be established.

The observed reduction in SERCA2a gene expression following exposure to CsA or KN-93 could have resulted from CsA or KN-93 mediated effects on calcium fluxes thereby indirectly affecting CN/CAMK-II pathway activity through an upstream mechanism. Upstream modulation of CN-NFAT signaling has recently been reported for cGMP-dependent protein kinase, which suppressed NFAT activation through reduction of L-type calcium channel activity [39]. Effects of CsA or KN-93 on calcium fluxes have so far not been reported and therefore render such an upstream modulation unlikely. Furthermore, the use of the "chemical phosphatase" BDM as a cross-bridge uncoupler has certain limitations. Several studies describe that in addition to its effects on force generation, BDM modulates the activity of several proteins including the L-type calcium channel and gap junction channels, possibly due to dephosphorylation [40–42], and effects on transcription regulation have also been suggested. The reported effects of BDM are however observed at higher concentrations (between 15 and 50 mM) than those required to inhibit contractile activity. The BDM concentration (7.5 mM) used in the present study is reported to maintain calcium transients [43] which was confirmed by our own observations (A. Muller, unpublished results).


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
References
 
In contraction-arrested NVCM, calcium-dependent signaling stimulates SERCA2a transcription. Both CN and CAMK-II pathways are involved in the calcium-dependent upregulation, most likely due to synergistic stimulation of the SERCA2a promoter by NFATc4 and MEF2c. Contractile activity and calcium-dependent signaling exert their actions on SERCA2a gene expression through distinct and independent pathways. Future studies should therefore address not only calcium-dependent but also mechanical-stress sensitive signaling by which contractile activity inhibits SERCA2a expression.


   Notes
 
Time for primary review 23 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 

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  6. Aoyagi T, Yonekura K, Eto Y, Matsumoto A, Yokoyama I, Sugiura S, et al. The sarcoplasmic reticulum Ca2+-ATPase (SERCA2) gene promoter activity is decreased in response to severe left ventricular pressure-overload hypertrophy in rat hearts. J. Mol. Cell. Cardiol. (1999) 31:919–926.[CrossRef][Web of Science][Medline]
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