Cardiovascular Research

Cardiovascular Research 1999 43(2):426-436; doi:10.1016/S0008-6363(99)00120-0
© 1999 by European Society of Cardiology

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Copyright © 1999, European Society of Cardiology

Sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA2) gene products are regulated post-transcriptionally during rat cardiac development

Antoine Ribadeau-Dumas^a, Marc Brady^b, Samuel Y Boateng^b, Ketty Schwartz^a and Kenneth R Boheler^b,c,*

^aUnité INSERM UR 153, Institut de Myologie, Groupe Hospitalier Pitié-Salpétrière, Paris, France
^bImperial College of Science, Technology and Medicine, National Heart and Lung Institute, London, UK
^cNational Institute of Health, National Institute on Aging, Baltimore, USA

^* Corresponding author. Tel.: +1-410-558-8095; fax: +1-410-558-8150 bohelerR{at}gre.nia.nih.gov

Received 26 November 1998; accepted 25 February 1999

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: The Sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA2) plays a major role in the contraction–relaxation cycle and is responsible for transporting calcium into the lumen of the sarcoplasmic reticulum. This study was performed to determine if the increase in SERCA2 messenger RNA (mRNA) abundance during the perinatal period is regulated transcriptionally. Methods: Transcriptional activity was determined by nuclear run-on assays and mRNA and protein abundances were determined during late fetal and early neonatal cardiac development in rat. Results: From nuclear run-on assays, SERCA2 gene transcription at 17/18 embryonic days (139±41 parts per million (ppm), n=7) did not differ from that at 20 neonatal days (139±37 ppm, n=6) after birth. No increase in transcriptional activity could be demonstrated during the time frame examined. In contrast, both alpha and beta myosin heavy chains showed significant changes in measured transcriptional activity. SERCA2 mRNA normalized to 18S RNA levels are very low in the fetus (9.8±1.9 to 13.4±4.9 arbitrary units (A.U.) from 17/18 to 19/20 embryonic days) and significantly increase from birth (15±3.8 A.U.) to reach a maximum at 20 days of age (29.1±9.5 to 48.3±7.0 in 15 to 20 neonatal days rats respectively). Similarly, SR Ca²⁺-ATPase protein levels are less abundant in the fetus (0.82±0.08 to 1.13±0.13 A.U./µg total protein) and reach a maximum at 15–20 neonatal days (3.08±0.58 to 2.98±0.17). Ca²⁺ uptake in the fetal heart is about one sixth the level seen in the adult, reaches the highest observed value at 5 days after birth (6.05±0.77 pmole Ca²⁺per µg/min) and remains relatively constant over the next 15 days. The activity increases even though phospholamban protein increases in abundance. Conclusions: Since the transcriptional activity of this gene is unchanged whereas the mRNA, protein abundance and activity increase, we conclude that the abundance of SERCA2 gene products is regulated primarily through post-transcriptional mechanisms during the perinatal period.

KEYWORDS Sarco(endo)plasmic reticulum Ca²⁺-ATPase; Heart development; Rat; Gene transcription; RNA

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See Editorial of this article by M.J.B. van den Hoff and A.F.M. Moorman (pages 288–290) in this issue.

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Myocardial function changes during ontogeny [1,2] and some of the developmental changes in the mechanical properties of the heart have been hypothesized to result from alterations in the abundance of proteins associated with the sarcoplasmic reticulum (SR). The SR controls myocardial contraction and relaxation through its regulation of cytosolic calcium concentrations [3]. Contraction is stimulated by calcium release from the SR through a calcium release channel. Cardiac relaxation occurs following removal of calcium from the cytosol primarily to the lumen of the SR through an ATP-dependent calcium transporter, the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA). Three genes and six isoforms of this transporter have been identified, SERCA 1, 2 and 3 [4]. The cardiac isoform, SERCA2a, is obtained by alternative splicing of SERCA2 gene transcripts [5,6]. Because of its high capacity to remove Ca²⁺ from the cytoplasm, the Ca²⁺-ATPase is the primary protein responsible for cardiac relaxation [7,8]. In fact, depending upon the species, the Ca²⁺-ATPase removes from 60 to 80% of all Ca²⁺ from the cytoplasm during end-systole and diastole. Ca²⁺-ATPase activity is regulated by phospholamban (PLB) [9]. In the nonphosphorylated form, PLB acts as an inhibitor of SERCA; when phosphorylated, PLB inhibition is relieved permitting SR Ca²⁺-ATPase activity to increase. In addition to phosphorylation, the levels of expression of PLB in relation to SERCA appear to affect the speed of myocardial relaxation [10].

Several studies have shown that SR function (calcium uptake and the calcium ATPase activity) during cardiac development is less pronounced in fetal than in adult rats [4,11–13]. This increase in function has been associated with an increased abundance of SERCA2 transcripts in more mature rat hearts [13–15]. We previously reported that SERCA2 transcripts are already abundant in the cardiogenic plate at nine embryonic days of rat development, and that the transcripts increase in abundance during late fetal development [15]. Several other groups have shown that the mRNA abundance increases significantly at birth, and protein abundance and activity are much higher in adult compared to fetal cardiac muscle [12,13,16,17]. Komuro et al. indicated that the enhanced SR Ca²⁺-ATPase protein abundance and increased SR Ca²⁺ uptake roughly paralleled, suggesting that the SERCA2 gene expression was regulated through pre-translational mechanisms. Since rat SERCA2 promoters can be activated by thyroid hormone in vitro [18–20] and since circulating thyroid hormone levels increase just before birth, it has been generally assumed that the increase in SERCA transcripts was due to transcriptional activation before birth. The recent description of significant developmental changes in DNA, RNA and protein abundance throughout the perinatal period have however complicated the interpretation of these published observations [21]. To test the hypothesis that SERCA2 gene transcription during the perinatal period would be activated prior to any increase in SERCA2 mRNA and protein levels, the present study utilizing nuclear run-on assays was performed and the data have been examined taking into account changes in DNA, RNA and protein abundance.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Animals
Wistar rats including 17–20-embryonic day (ED) fetuses, 1, 5, 15, 20 neonatal day (ND) and 2-month-old adults unless otherwise indicated were used in this study. Hearts were collected from each age group and were dissected free of atrial tissue and large blood vessels before nuclei isolation, crude homogenates preparation and freezing for mRNA measurements.

2.2 Isolation of myocardial nuclei and in vitro transcription
2.2.1 Isolation of myocardial nuclei
Total cardiac nuclei were isolated as previously described [22,23]. Prenatally, whole hearts were obtained from fetuses (17–20 ED) of three to seven pregnant rats (25 to 35 pooled animals), and post-natally, ventricles were obtained from five to 20 rat pups (1–20 ND). All steps in the procedure were performed at 4°C. After rinsing in saline, 0.75–1.0 g of tissue was homogenized in buffer NA (300 mM sucrose, 10 mM Tris pH 8.0, 2.5 mM Mg²⁺–acetate, 0.5 mM dithiothreitol (DTT), 0.25% Triton X-100, 0.1 mM phenylmethylsulfonylfluoride (PMSF), 15 units/ml recombinant RNasin (Promega Biotech) with 5–10 strokes of a Teflon homogenizer. The homogenate was filtered through a nylon membrane, and buffer NB (2.4 M sucrose, 10 mM Tris pH 8.0, 2.5 mM Mg²⁺–acetate, 0.5 mM DTT 0.1% Triton X-100, 0.1 mM PMSF, 15 units/ml rRNasin) added. This solution was layered onto a cushion of buffer NB and centrifuged at 25 000 rpm for 1 h at 4°C in a SW28 Beckman rotor. The pellet was resuspended in NA without triton X-100, centrifuged at 2000 g for 20 min, the supernatant discarded and the washed pellet resuspended in 1.5 ml of NA buffer (without Triton X-100). The number of nuclei were determined, followed by centrifugation at 5000 rpm for 5 min, and the pellet was resuspended to give a final concentration of not greater than 0.5–1.0x10⁷ nuclei per 200 µl Keller storage buffer. The nuclei were frozen in liquid nitrogen and stored at –80°C.

2.2.2 In vitro transcription
Nuclear transcription assays and hybridizations were performed as previously described [22,23]. Briefly, isolated nuclei (5x10⁶ to 1x10⁷) were resuspended in the reaction mix (40 mM Tris–HCl pH 8.3, 150 mM NH₄Cl, 7.5 mM MgCl₂, 0.625 mM ATP, 0.313 mM each of GTP and CTP, and 0.5 mCi [³²P]UTP (800 Ci/mmol, Du Pont), 1200 units/ml rRNasin), and incubated at 27°C for 20 min. Radionucleotide incorporation was determined by DE-81 filter washes. After a 20-min incubation period, the reaction was stopped by addition of 75 units RQ1 DNase (Promega) and 40 units of rRNasin and incubated at 27°C for 15 min. The reaction was then centrifuged for 2 min at 5000 rpm in an Eppendorf microcentrifuge, and the neonascent transcripts isolated as previously described [23].

A BioDot SF (BioRad Laboratories) apparatus was used to prepare the slot blots containing the denatured plasmid pSERCA6 [24], fibronectin, alpha and beta myosin heavy chain [22] and as control the plasmid pBluescript (Stratagene). In some control experiments, single-stranded pRH39 derived from M13 subclones was used as positive and negative controls. Five micrograms of each plasmid were made up to a final volume of 50 µl with water and 3 µl of 5 M NaOH added. This was incubated at 95°C for 10 min and allowed to cool for 5 min. In the case of single-stranded M13 derived clones, only heat was used to get rid of any secondary structure. Fifty microliters of 4 M ammonium acetate was added, vortexed and 50 µl were added to each slot of hybond N+ nylon membranes (Amersham). Upon addition, the slot was rinsed two times with 50 µl of 1 M ammonium acetate and then rinsed with 2X SSC. The membrane was dried and UV fixed with a Stratalinker (Stratagene). Probe annealing to the nylon membranes containing specific sequences for SERCA was performed by addition of 5x10⁵ cpm/ml labeled transcripts to 5–10 ml of a pre-hybridization solution containing 50% deionized formamide, 0.1% SDS, 5X SSC, 10 mM EDTA, 10 mM Tris–HCl, pH 7.5, 2.5X Denhardt’s solution, 40 mg/ml herring sperm DNA (Promega), 40 ng/ml poly A (Pharmacia) and 20 ng/ml poly G (Pharmacia) at 55°C for >40 h. Membrane washes were for 3 h as previously described [22] with the exception that the RNase wash was supplemented with 1.5 µl RNase T1 (100 units/ml, Boehringer) in addition to the 10 µl of RNase A per 200-ml wash solution. Hybridization efficiencies were as previously described [23,25].

2.3 Measurements of SERCA2 and MHC mRNA abundance
Total cellular RNA was prepared from heart tissues according to Chomczynski and Sacchi [26]. The amount of SERCA2 mRNA was determined by Northern and Dot-blot analysis as described [27,28]. RNA was transferred to nylon membranes (Hybond N+, Amersham), cross-linked, and hybridized with cDNA probes for rat SERCA2 [29] labeled with (-³²P) dATP by random priming (Megaprime Kit, Amersham) according to the manufacturer’s instructions. Hybridization was carried out at 42°C for >18 h in 50% deionized formamide, 1% SDS, 5X Denhardt’s solution, and 100µg/ml herring sperm DNA. Membranes were washed for successive 20-min periods under progressively more stringent conditions, terminating at 0.1X SSPE/0.1% SDS at 65°C for SERCA2. Membranes were exposed to Kodak XAR film at –70°C and developed after varying times from 1–4 days to ensure that signals were within the linear range for densitometric analysis. To normalize for possible differences in amount of RNA loading, membranes were subsequently dehybridized (0.1% SDS for 2x30 min, heated to 100°C) and hybridized (under the above conditions however omitting formamide) with (³²P)-end labeled oligonucleotide complementary to 18S ribosomal RNA (5' ACG GTA TCT GGA TCG TCT TCG AAC C 3'), washed (two times for 15 min at 3X SSPE/0.1% SDS at room temperature), and autoradiographed as previously described [30]. Autoradiograms were quantified using digital imaging densitometry and data are expressed as a ratio to 18S RNA. Total RNA content is expressed as a ratio between the total RNA isolated (µg) and the initial quantity of tissue (mg) used for the RNA extraction.

The relative abundance of alpha and beta MHC gene transcripts were performed using an RT–PCR based technique utilizing the forward (5'-GCA GAC CAT CAA GGA CCT-3') and reverse (5'-GTT GGC CTG TTC CTC CGC C-3') primers for amplification and the restriction enzyme Tru 9I to distinguish between the two isomRNAs. Experiments were exactly as described [31].

2.4 Measurements of SERCA2 splice variants
Northern blots were run as described above with 20 µg of total RNA isolated from a separate group of Wistar rats. RNA was transferred to Magna Transfer uncharged nylon membrane (Micron Separations, MA., USA) overnight at room temperature in 2X SSC. An EcoRI fragment of pSERCA6 was isolated and labelled as described above (probe A). A 113-bp probe specific for one 3' untranslated region splice variant [24] of SERCA2 was generated by PCR (Robocycler 40, Stratagene) using pSERCA6 as template, two primers [Serca 55 (5'-GCT TCT CAC AGT GCA TGT CTG ACT G-3') and Serca 56 (5'-ATC GTA GAA TCG ATT TAT CCT-3')] and Pfu Turbo DNA polymerase (Stratagene) in a volume of 50 µl containing Pfu buffer (200 mM Tris–HCl, pH 8.8, 100 mM KCl, 100 mM (NH₄)₂SO₄, 20 mM MgSO₄, 1% Triton X-100, 1 mg/ml nuclease-free serum albumin). The resulting PCR mixture was loaded onto a 1% agarose gel and the fragment was purified using Genelute Spin columns (Supelco, Sigma) and resuspended in TE, pH 8.0. Partial clonal analysis of the fragment was performed by restriction enzyme digestion to ensure fragment authenticity. The verified 113-bp fragment was double labelled (probe B). For this 25 ng of the 113-bp fragment, 2 µl of primer Serca 56 at 100 pmoles/µl, 1 µl of Klenow (2 U/µl), 5 µl of (³²P) dGTP and 5 µl (³²P) dCTP were incubated at 37°C for 10 min as described above.

Membranes were prehybridized for 60–90 min at 68°C in ExpressHyb solution (Clontech, California, USA). Probes were added for 1 h at 68°C at 1.5 million cpm/ml. The membranes were washed sequentially in 2XSSC/0.05% SDS for 10 min (4x) at room temperature, in 0.5% SSC/0.1% SDS for 20 min at 45°C, in 0.5% SSC/0.1% SDS for 15 min (x2) and at both 50°C and 60°C. Membranes hybridized with Probe A were washed as described above. The membrane was stripped between hybridizations with 5 mM Tris–HCl, pH 8.0, 0.2 mM EDTA, 0.05% sodium pyrophosphate, 0.1% Denhardts solution at 65°C for 60 min (x2) and subsequently washed in 1x SSPE and exposed to film overnight to ensure no residual radioactivity remained on the blot. Signals were determined by exposure to either film overnight (X-OMAT, Kodak) at –80°C or in an image cassette and quantified using the ImageQUanT system (Molecular Dynamics, SunnyVale, CA., USA). Data from two to four samples are expressed as a ratio between the signals obtained with the 113-bp probe and the nonspecific SERCA2 probe. No corrections were made for the exposure times.

2.5 SERCA2a and PLB protein analysis by Western blotting and quantification by ELISA
Western blotting was performed on protein extracts from total left ventricles in adult rats. Proteins were separated using SDS–polyacrylamide gel electrophoresis (SDS–PAGE) — 7.5% for SERCA2a and 15% for PLB — and transferred by electroblotting onto nitrocellulose membranes or PVDF membranes (Hybond C, Amersham; Immobilon, Millipore), respectively. Membranes were incubated with the appropriate antibodies: a rabbit polyclonal antibody specific for SERCA2a, generously provided by Dr F. Wuytack [32], or a mouse monoclonal specific antibody for PLB (Euromedex, France). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (a goat anti-rabbit or anti-mouse antibody). The proteins were visualized using the enhanced chemiluminescence system (SuperSignal, Pierce, Rockford, USA). Quantification of SERCA2a and PLB protein levels were performed using an Enzyme Linked ImmunoSorbent Assay (ELISA) developed in the laboratory. Briefly, twelve serial dilutions of total cardiac protein (starting with 20 µg) from the crude homogenates were adsorbed on microtiter plates (Immulon, Dynatech) using an adsorption solution (Na₂CO₃ 100 mM pH 9.6) overnight at 4°C. The microtiter plates were washed four times, for 30 s each with PBS–0.1% Tween 20, and incubated for 90 min at 37°C in the presence of the rabbit polyclonal primary antibody specific for SERCA2a (1/10 000 dilution) or for 2 h with the monoclonal antibody for PLB (1/10 000 dilution) in a 200 µl final volume. Four washes were performed, and the secondary antibodies — an alkaline phosphatase-conjugated goat anti-rabbit or anti-mouse antibody, were added to each well at a 1/10 000 dilution in a 150-µl final volume. Microtiter plates were incubated for 1 h at 37°C. After four washes, alkaline phosphatase activity was measured by incubating the microtiter plates — 1 h for SERCA2a and 2 h for PLB at 37°C — with the revelation solution (1 M diethanolamine, 12.3 mM MgCl₂, 38.5 mM NaN₃, pH 9.8). The absorbances were read using a microtiter plate spectrophotometer at the 405-nm wavelength. Total tissue protein concentration (in µg/µl) and quantity (µg) were determined using the BCA reagent assay (Pierce, Rockford, USA) with BSA as a standard. Total protein content is expressed as a ratio between the measured protein quantity (mg) and the starting quantity of tissue (mg).

2.6 Measurement of K⁺-oxalate stimulated Ca²⁺-uptake in crude homogenates
One hundred to 200 milligrams of freshly pooled ventricles (from 17/18 embryonic days to 5 days of age) or isolated left ventricles (from 10 days of age to adults) were homogenized in a medium containing 30% glycerol, 20 mM Hepes pH 7.40, 5 mM NaN₃, 1 mM PMSF, 5 mM pyrophosphate and 50 mM sodium fluoride using a polytron homogenizer (PTA 20TS, Kinematika, Switzerland) for 3–5 s bursts at setting 5. Protein concentration in the homogenates was determined as described above. Measurement of Ca²⁺-uptake was performed as described [33]. After 5 min of incubation, 0.5-ml aliquots were filtered through millipore filters (HAWP 0.45 µm, Bedford, Massachussets) by aspiration and immediately washed with 15 ml of ice-cold 100 mM KCl, 30 mM MOPS, 1 mM EGTA and 10 mM histidine, pH 6.85. The radioactivity trapped on the filter was counted in a liquid scintillation counter (Beckman Instruments, Fullerton, California). The amount of the calcium trapped in the SR was determined from the averages of quadruplicate measurements and expressed as pmoles of Ca²⁺ per µg of total protein per minute. All data are expressed as a percentage of the maximum values.

2.7 Statistical analysis
Values are expressed as mean±SEM. Statistical assessment of mean value comparisons for developmental stages were performed by a Student’s t-test. When conditions of parametrical t-tests were not satisfied, an ANOVA followed by nonparametrical analyses (Kruskal–Wallis or Mann–Whitney tests) were used to examine the effects of development on changes in transcription, mRNA abundance, protein levels and protein activity. P values of <0.05 were considered significant.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Gene transcription during cardiac development in the rat
Total (³²P) UTP incorporation in cardiac nuclei was rapid and plateaued 20 min after initiation of the reaction (Fig. 1). The radioactive incorporation did not differ between any of the groups examined and maximum incorporation averaged 0.60±0.22 cpm per nucleus and ranged from 0.50±0.20 to 0.69±0.17 cpm per nucleus. This incorporation was about three-fold greater that that seen in adult nuclei (0.24 cpm per nucleus, greater than three months). Radioactively labeled endogenous nuclear RNAs were isolated from the run-on reactions and hybridized to membranes containing probes specific for several cardiac mRNAs (Fig. 2A). Alpha and beta MHC were both transcriptionally regulated during the perinatal period (Table 1) — alpha MHC was significantly upregulated, whereas, beta MHC was down-regulated. Fibronectin (FN) transcription was generally not detectable, however signals above background were measured in four out of 16 of the run-on assays performed with nuclei isolated from fetal animals (17/18 ED).

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Total mean radioactive incorporation measured in nuclear run on assays. Radioactive incorporation was measured (between 0 and 20 min) in isolated nuclei from hearts at 17/18 ED, 19/20 ED, 1 ND, 5 ND, 15 ND and 20 ND (n=4 for each). For each developmental stage, nonspecific signals at time t0 (considered as zero) were substracted from the other time points (0, 5, 10 and 20 min). Results are expressed as cpm per nucleus.

 

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 (A) Nuclear run-on assays. Image derived from an autoradiogram showing the extent of hybridization of purified transcripts with the plasmids pSERCA6, alpha MHC and M13 derived single-stranded beta MHC, fibronectin (FN) and pBluescript (pBS). Nuclear run-on assays were performed using between 5 and 10 million nuclei as described in the Methods section. Data are shown from a single set of experiments with nuclei isolated from 17 ED, 1 ND, 5 ND and 20 ND rat hearts. In this experiment, 500 000 cpm/ml were hybridized to denatured plasmids covalently linked to nylon membranes. (B) Synthesis of SERCA2 transcripts as a function of age. All data have been expressed in parts per million (ppm) that have been corrected for hybridization efficiencies. The reported values thus reflect the transcriptional activity of specific genes in isolated cardiac nuclei. Statistical significance was determined by a nonparametrical Mann–Whitney test (U-test). Values are expressed±SEM. There were no significant differences between any of the time points considered.

 

View this table: [in this window] [in a new window]   Table 1 Nuclear run-on assays: -MHC and β-MHC gene transcriptiona

 
In cardiac nuclei, no significant difference in SERCA2 transcriptional activity could be demonstrated between any of the time points examined (Fig. 2B). The highest individual level of expression was seen in the 17/18 ED rat hearts but this value was almost identical to that seen at 20 ED. The least incorporation in parts per million (ppm) was measured in 1-neonatal-day-old animals, but because of the range of values measured in the nuclei preps, no significant difference with 17/18 ED or 20 ND could be demonstrated. On membranes with single-stranded probes generated from M13 and specific for sense and antisense SERCA2 mRNA sequences, positive signals were only seen when hybridized with antisense probes, excluding the possibility of nonspecific signals (data not shown). No significant difference could be demonstrated between any of the time points examined, indicating that SERCA2 transcription is in a relatively steady-state situation throughout the perinatal period (P>0.05 compared to all other time points measured).

3.2 SERCA2a mRNA levels during cardiac development in the rat
In late fetal (17/18 and 19/20 ED) and 1 ND rat hearts, SERCA2 mRNA abundance increased, but did not significantly differ [arbitrary units: 9.8±1.9 (n=8), 13.4±4.9 (n=4) and 15.0±3.8 (n=8), respectively](Fig. 3A). SERCA2 transcripts significantly increased at five neonatal days and showed maximal amounts at 20 days after birth (Fig. 3B). At two months of age, the SERCA2 transcripts were significantly more abundant (P<0.05) than that seen in late fetal and newborn rat hearts. Alpha MHC transcripts increased significantly and beta MHC transcripts decreased significantly during the perinatal period (P<0.05) (data not shown).

View larger version (34K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 (A) Dot blot of SERCA2 and 18S gene expression. The SERCA2 image was generated from a phosphoimager, whereas the 18S image was prepared by autoradiography. Analyses were performed using both imaging techniques. Hybridizations were also performed on Northern blots to ensure accuracy of the results. (B) Relative amounts of SERCA2 transcript abundance with age as a function of total RNA (18S RNA). Statistical significance was determined by a nonparametrical Mann–Whitney test (U-test). Values are expressed±SEM. P<0.05 was considered significant.

 
During the time period examined, total RNA yields isolated from each of the age groups did not differ markedly as a function of age (Table 2). From 17 ED to 1 ND, the amounts of total RNA isolated averaged 2.5 µg/mg wet weight. Post-natally, total RNA per mg tissue wet weight decreased by 30% to an average of 1.7 µg/mg. During this same period, SERCA2 mRNA content increased by 5-fold as a function of 18S ribosomal RNA, but if looked at on a per mg basis as in van den Hoff et al., [21] the increase was only two- to three-fold (Table 2).

View this table: [in this window] [in a new window]   Table 2 Relative expression of SERCA2 gene products during rat cardiac developmenta

 
3.3 Quantification of the SERCA2a protein levels by ELISA
The anti-SERCA2a antibody was tested on total protein extracts by Western Blotting to ensure specificity. One clear single band of the expected molecular mass (110 kDa) was seen (Fig. 4A). This antibody was used in an ELISA assay for quantification of the SR Ca²⁺-ATPase 2a isoform. Results are shown in Fig. 5. In late fetal rat hearts, SERCA2a protein levels were significantly lower compared to those measured in 15- and 20-day-old rats (0.82±0.08 and 1.13±0.13 vs. 3.08±0.58 and 2.98±0.17, P<0.05). It was significantly increased in neonates (P<0.05 vs. 17/18 ED), remained constant in 5- and 10-day-old rats, and increased to the highest value at 15 and 20 neonatal days. At two months of age, SERCA2a protein levels were significantly decreased compared to 20 ND. The amount of SERCA2a protein and total wet weight cardiac mass both increased by nearly three- to four-fold (Table 2). This resulted in a net 11-fold increase of SERCA2a protein during the perinatal period when analysed as by van den Hoff et al. [21]

View larger version (29K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Western blot analysis of SERCA2a and PLB. (A) SERCA2a: 2.5, 5 and 10 micrograms of total protein homogenates were separated on 7.5% SDS–PAGE gels. Nitrocellulose membranes were probed with the SERCA2a specific polyclonal antibody as described in methods. A single band of 110 kda was observed. (B) PLB: 25 µg of total protein from crude homogenates (1) and from SDS extracted proteins (2) were separated on 15% SDS–PAGE gels. After transfer, PVDF membranes were probed with a PLB specific monoclonal antibody (Euromedex) as described in Methods. Results from these Westerns showed a single band in SDS–protein extracts (without phosphatase inhibitors), and at least two bands in crude homogenates (prepared with phosphatase inhibitors). The upper band corresponds to a phosphorylated form of phospholamban (lane 1).

 

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Quantification of SERCA2a protein abundance during cardiac development. The amount of SERCA2a protein was determined by ELISA technique in crude homogenates using the antibodies described in Fig. 4. The 1/2 absorbance at 405 nm was normalized by the corresponding amount of total protein used for the measurement. Statistical significance was determined by a nonparametrical Mann–Whitney test (U-test). Values are expressed±SEM. P<0.05 was considered significant.

 
3.4 Rate of calcium uptake during cardiac development in the rat
The rate of Ca²⁺-uptake in fetal stages (0.55±0.28 pmoles Ca²⁺ per µg/min and 0.93±0.49 pmoles Ca²⁺per µg/min in 17/18 and 19/20 ED, respectively) was only about 10–15% of that seen in 5–20 neonatal day animals (Fig. 6 and Table 2). It was significantly increased by four to six-fold in one day old neonates and, from 5 to 20 neonatal days, the rate of calcium uptake plateaued. The yield of total cardiac protein (Table 2) was however significantly lower in the fetus than in the neonates (32.1±4.9 µg/mg vs. 84.6±9.5 µg/mg, P<0.05). Maximal protein yields were obtained in crude homogenates from 5-day-old rat hearts (143.9±16.3 µg/mg, P<0.05 vs. 17/18 ED). The increase in uptake was earlier and greater than the increase in protein yield, indicating a maturation of the sarcoplasmic reticulum and enhanced SR Ca²⁺-ATPase function.

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Measurement of oxalate-supported Ca2+-uptake by the SR in crude homogenates during cardiac development. Addition of calcium was used to initiate each reaction. Each experiment was performed a minimum of four times on each homogenate in either the presence or absence of ATP. Calcium uptake was linear over a period of 6 to 8 min and plateaued between 10 and 15 min. In the absence of ATP or calcium, uptake was not detectable. The calcium uptake measured in the crude homogenates was completely inhibited by addition of 100 nM thapsigargin. No nonspecific uptake above background levels (no ATP) could be demonstrated, excluding the possibility that other mechanisms were involved in the uptake of calcium in these homogenates. The calcium precipitations measured under these experimental conditions were therefore the result only of endogenous SR Ca2+-ATPase activity. Statistical significance was determined by a non parametrical Mann–Whitney test (U-test). Values are expressed±SEM. P<0.05 was considered significant.

 
3.5 Potential mechanisms
3.5.1 SERCA2 splicing variants
The relative abundance of two cardiac splicing variant of SERCA2 mRNA was determined by Northern blotting. The difference between the two variants consisted of a 128 nucleotide sequence that was either present (isoform A) or absent (isoform B) in the 3' untranslated region of mature SERCA2a transcripts. Both isoforms of SERCA2 mRNA showed an increase in abundance throughout perinatal development. As a ratio of Form B to Form A (signal isoform B/signal isoform A), no change in the relative abundance of the splicing variant to total SERCA2 could be demonstrated from 20 ED to adult; however, in the 18 ED rats, isoform B was 60% more abundant. Ratiometric signal data of isoform B to isoform A are as follows: Adult, 0.34; 20 ND, 0.34; 10 ND, 0.34; 5 ND, 0.33; 1 ND, 0.35; 20 ED, 0.33; 18 ED, 0.54. No signals were obtained from liver, excluding the possibility of nonspecific hybridization signals (data not shown).

3.5.2 PLB protein determination
Depending upon the method of preparation, Western blot analysis indicated the presence of either one or multiple phosphorylated forms of PLB (Fig. 4B). All ELISA assays were however performed using the same antibody and the results are shown in Fig. 7. In late fetal rat hearts (17/18 and 19/20 ED), PLB protein levels were significantly lower when compared to 20 neonatal-day-old rats (0.164±0.014 and 0.338±0.022 vs. 0.578±0.075, P<0.05). PLB quantities were significantly elevated in all post-natal samples relative to 17/18 ED fetal rat hearts (P<0.05).

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Quantification of PLB protein abundance during cardiac development. The amount of PLB protein was determined by the ELISA technique in crude homogenates using the antibodies described in Fig. 4. The 1/2 absorbance at 405 nm was normalized by the corresponding amount of total proteins used for each measurement. Statistical significance was determined by a nonparametrical Mann–Whitney test (U-test). Values are expressed±SEM. P<0.05 was considered significant.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The nuclear run-on assay employs nuclei which retain endogenous RNA polymerase activity, without measureable RNase or DNase activities and which has the capacity to transcribe in vitro in a tissue-specific and DNA strand-specific manner [22]. The nuclear run-on assay thus represents a system that is as close to the intact cell as possible. During the perinatal period, -MHC and β-MHC genes were transcriptionally regulated, transcribed in a tissue-restricted manner and the results described here from 20 day old rat hearts were consistant with what we previously reported at 23–24 neonatal days [22]. In fetal nuclei, the transcriptional activity of fibronectin (FN) was detectable in four of 16 samples. It is impossible to determine what percentage of fetal nuclei were myocytic, but the fact that FN was only weakly transcribed whereas both - and β-MHC genes were transcribed in abundance argues for a predominance of myocytic nuclei.

The major finding in this study is that quantifiable SERCA2 gene transcription remains relatively constant from 17/18 embryonic days to 20 days after birth. This gene product is transcribed in a DNA strand-specific manner (M13 control experiments, data not shown) and its hybridization to pSERCA6 is template specific. SERCA2 mRNA transcripts and SR Ca²⁺-ATPase protein abundance and function increased significantly after birth. These data argue strongly for post-transcriptional and significant post-translational regulation of SERCA2 gene products during the perinatal period.

The mechanism through which SERCA2 transcript abundance increases during the perinatal period is unclear. SERCA2 transcripts are present in rat at 9 ED and the signal intensity for this mRNA increases with fetal development [15]. These results suggest that the SERCA2 gene transcription is activated very early in cardiac development. We cannot exclude the possibility that SERCA2 gene transcription is reactivated before our first measured time-point at 17 EDs. If reactivation of transcription were to occur before 17 ED, then one would need to postulate a post-transcriptional mechanism responsible for degrading SERCA2 transcripts in late fetal myocardium and which ceased to be a factor post-natally. Alternatively, if a significant contribution of SERCA2 gene transcription during the fetal stages were of non-myocytic origin, then it is possible that we have overestimated SERCA2 transcription in myocytes before birth. If true, any potential and modest increase in SERCA2 gene would still not account for the very significant increases in mRNA, protein and function seen post-natally.

Recently we described a 3' splicing variant of SERCA2a transcripts in heart (isoform B) which had been described in other tissues [24]. We speculated that alternative splicing of the 3' region of SERCA2 transcripts might play a role in its stability. The abundance of this splicing variant relative to total SERCA2 mRNA abundance did not change between 20 ED and 20 ND or 2-month-old adults. Importantly, this variant was much more abundant in 18 ED rat hearts. In fact, its presence appears to be almost three-fold greater at 16 ED than at 18 ED (n=2, unpublished data MB). These data suggest that this 128-nucleotide splice variant of mature SERCA2 transcripts is regulated developmentally, and when abundantly present, might play a role in destabilizing SERCA2 mRNA. From computer analyses, we have been unable to identify any part of this 128-nucleotide sequence that has a known function in mRNA stability (e.g. AUUUA) [34]. Further studies will therefore be necessary to address this possibility, particularly since these analyses were performed on a separate group of rats than that used in the run-on assays.

Total RNA and protein concentration and content are known to show developmental changes that closely match growth phases of the developing heart [21]. When we report the data as a function of cardiac mass, an increase in SERCA2 mRNA is measurable and significant, albeit to a lesser extent (Table 2) than that seen normalized to total RNA (18S RNA). Total SERCA2 protein content also increases, but in terms of concentration, the increase is proportional to the increase in cardiac mass. The functional gains in Ca²⁺-ATPase activity were also clearly greater than the increase in protein. It is unclear whether total content or concentration of mRNA or protein is more appropriate, as an index of expression, particularly when considered in light of the dramatic functional changes seen throughout the perinatal period. What is remarkable however, is that in the absence of measurable transcriptional activation, SERCA2 mRNA and protein content increase dramatically. The increase in terms of concentration is less but still strongly supportive of the argument for post-transcriptional regulation of SERCA2 gene product abundance.

The enhanced Ca²⁺ uptake in the homogenates shows that post-translational mechanisms are predominant features of the functional gain seen during the perinatal period. As SR Ca²⁺-ATPase activity is regulated by PLB, its expression was examined. SR Ca²⁺-ATPase and PLB protein levels were found to increase in parallel, both showing maximal abundance at 15 or 20 postnatal days, and suggesting that PLB mediated inhibition of the SR Ca²⁺-ATPase should occur post-natally. The lack of increased oxalate-stimulated calcium uptake late in perinatal development argues for an enhanced role of phospholamban. Its role would however depend on its phosphorylation state. Preliminary experiments (unpublished observations, A.R.-D.) did not detect any change in the phosphorylation state of PLB suggesting that the ratio of SR Ca²⁺-ATPase to PLB and not the phosphorylation state of PLB was an important determinant of Ca²⁺-ATPase activity throughout fetal and neonatal development. In mouse heart, this ratio has already been shown to affect SR function [35–37] and is consistent with the results presented here.

Other levels of regulation should be considered. In severe experimental cardiac hypertrophy, SERCA2 transcription decreases [38] concomitant with a decrease in the concentration of the mRNA and protein. This implies that in some instances the regulation of SERCA2 transcription can be the major regulatory point. In transgenic mice with multiple copies of the rat SERCA2 cDNA and high SERCA2 mRNA expression (160% increased) however, the transgene protein expression increased by only 20%. It was suggested that differences between the increased SERCA2a mRNA and relative protein synthesis rate might have resulted, in part, from the efficiency of translation of the SERCA2 transgene derived mRNA [39]. The exact mechanisms responsible for this discordance between RNA and protein are unclear, but there is ample evidence in other systems supportive of both transcriptional and post-transcriptional regulatory mechanisms for individual gene products [40,41].

In conclusion, SR Ca²⁺-ATPase mRNA and protein abundance increase in the absence of transcriptional activation during the perinatal period, potentially through stabilization of mRNA transcripts. SR Ca²⁺-ATPase protein follows changes in its mRNA abundance, but higher levels compared to its mRNA content (three - to four-fold higher) suggest that the SERCA2 gene products are also regulated to a significant extent at a translational and/or post-translational level. Clearly, multiple factors are important. The implication of these data are not of immediate consequence in the clinic, but are highly supportive of our earlier conclusions [42] that in both normal and disease states, measurements of SERCA2 mRNA in heart should not be used as an indicator of SR Ca²⁺-ATPase protein expression or function. Identification and precise characterization of the factors (proteins) responsible for these phenomena, will be essential for a clear understanding of how the transcriptional and post-transcriptional mechanisms regulating SERCA2 gene expression affect normal and abnormal cardiac growth.

Time for primary review 26 days.

	Acknowledgements

 
This work was supported by the British Heart Foundation (PG/93148), the Institut National de la Santé et de la Recherche Médicale (INSERM), the Association Française contre les Myopathies (AFM), Glaxo-Wellcome (Dr. Jorge Kirilovsky) and the NIH/NIA. A. Ribadeau-Dumas was a recipient of a fellowship from the AFM. M. Brady was supported by a BHF studentship. We would like to thank Maren Koban, Dr. Marc Y. Fiszman, Dr. Lucie Carrier, and Dr. Gisèle Bonne, for helpful discussions and for critical readings of the manuscript. We also thank Mrs. Claudine Wisnewsky and Mr. John Hutchinson for technical assistance with the animals.

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