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Am J Physiol Heart Circ Physiol 291: H1101-H1108, 2006. First published March 3, 2006; doi:10.1152/ajpheart.00660.2005
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Regulatory role of ovarian sex hormones in calcium uptake activity of cardiac sarcoplasmic reticulum

Tepmanas Bupha-Intr and Jonggonnee Wattanapermpool

Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand

Submitted 20 June 2005 ; accepted in final form 1 March 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Alterations in the intracellular Ca2+ handling in cardiomyocytes may underlie the cardiac dysfunction observed in the ovarian sex hormone-deprived condition. To test the hypothesis that ovarian sex hormones had a significant role in the cardiac intracellular Ca2+ mobilization, the sarcoplasmic reticulum (SR) Ca2+ uptake and SR Ca2+-ATPase (SERCA) activity were determined in 10-wk ovariectomized rat hearts. With the use of left ventricular homogenate preparations, a significant suppression of maximum SR Ca2+ uptake activity, but with an increase in SR Ca2+ responsiveness, was demonstrated in ovariectomized hearts. In parallel measurements of SERCA activity in SR-enriched membrane preparations from ovariectomized hearts, a suppressed maximum SERCA activity with a leftward shift in the relationship between pCa (-log molar free Ca2+ concentration) and SERCA activity was also detected. A significant downregulation of SERCA proteins and reduction in the SERCA mRNA level were observed in association with suppressed maximum SERCA activity. While there were no changes in total phospholamban and phosphorylated Ser16 phospholamban levels, a decrease in phosphorylated Thr17 phospholamban as well as an increase in the suprainhibitory, monomeric form of phospholamban stoichiometry was found. Estrogen and progesterone supplementations were equally effective in preventing changes in ovariectomized hearts. Our data showed for the first time that female sex hormones played an important role in the regulation of the cardiac SR Ca2+ uptake. Under hormone-deficient conditions, there was an adaptive response of SERCA that escaped the regulatory effect of phospholamban.

sarcoplasmic reticulum Ca2+-adenosine 5'-triphosphate activity; phospholamban; phosphorylation

THE CARDIOREGULATORY ROLE of female sex hormones in contractile activity has been raised in many studies (23, 24, 28, 34–36). Expression of both estrogen and progesterone receptors in myocytes indicated a direct effect of the hormones on myocardial function (14, 18). Studies of postmenopausal women using echocardiography have demonstrated significant decreases in cardiac contractility compared with those of premenopausal women (23, 24). Reductions in stroke work and fractional shortening have also been demonstrated in studies using whole heart preparations of ovariectomized rats (28). In addition, suppression of both myosin ATPase activity and maximum myofibrillar ATPase activity with an upregulation of beta-myosin heavy chain in ovariectomized rat hearts was reported (28, 34, 35). All evidence indicated important roles of female sex hormones in myocardial activation. The striking finding was an increase in the Ca2+ responsiveness of cardiac myofilament activation with almost the same magnitude in ovariectomized hearts as that in cardiomyopathic failing hearts (17, 34, 35, 38, 39). The information thus implied a potential of cardiomyopathic induction by female sex hormone deficiency. It was, however, not known how myofilament Ca2+ hypersensitivity was induced in the hormone-deficient heart. A reasonable hypothesis was that this effect may be an adaptive response of the heart to changes in the intracellular free Ca2+ availability induced by ovariectomy.

Changes in the intracellular Ca2+ homeostasis possibly led to cardiac myofilament alteration and subsequent adaptation in ovariectomized hearts. This hypothesis was also intended for failing hearts, in which defective intracellular Ca2+ handling has been reported (1, 6, 22). Reduction in the Ca2+ transient amplitude with prolongation of Ca2+ decay was typically demonstrated in isolated cardiac myocytes of both human and animal failing hearts (1, 6, 22). Among the three major Ca2+ handling proteins, sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA), ryanodine receptor calcium release channel, and Na+-Ca2+ exchanger, SERCA was most the likely to play a significant role in the adaptive changes. The decreased peak systolic Ca2+ and prolonged Ca2+ sequestration in failing hearts were remarkably associated with reductions in SR Ca2+ uptake activity and SERCA expression (6, 10). Moreover, the suppressed SERCA activity was found to result from the increased inhibitory control by the dephosphorylated phospholamban (3, 10). Indeed, this body of evidence suggested a possible hypersensitive adaptation to the suppressed SR Ca2+ availability in the failing myocardial activation. On the other hand, the regulatory role of female sex hormones in cardiac intracellular Ca2+ mobilization has been much less extensively studied. Despite the controversial data in diastolic Ca2+ level and Ca2+ transient amplitude, a prolongation of Ca2+ decay observed in failing hearts was also reported in the ovariectomized cardiomyocytes (25). Therefore, adaptive alterations in SR Ca2+ removal activity associated with the regulatory role of female sex hormones were examined.

To address whether the ovarian sex hormones played a cardioregulatory role in SR Ca2+ uptake activity, we measured SR Ca2+ uptake and SERCA activities in 10-wk ovariectomized rat hearts. Alterations in the regulatory protein of SERCA activity were also analyzed. Our results demonstrated that deprivation of ovarian sex hormones induced a decrease in the cardiac SR Ca2+ uptake activity through suppression of both the activity and the expression of the SERCA pump. The suppressed uptake activity was due, in part, to an increased interaction of SERCA with the nonphosphorylated phospholamban proteins. In addition, estrogen and progesterone equally exerted a regulatory effect on SR Ca2+ uptake function.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal preparation. Female Sprague-Dawley rats weighing between 180 and 200 g (8–9 wk old) were sham operated or ovariectomized as previously described (34). Ovariectomized rats were randomly divided into control and hormone-supplemented groups. Deficiency of ovarian sex hormones was verified by the reduced uterine weight after the animals were killed. The rats were individually housed in an 8 in. x 10 in. hanging cage and given rat chow and water ad libitum. Two days after the operation, hormone supplementation was started by subcutaneous injection of estrogen (5 µg/rat), progesterone (1 mg/rat), or estrogen plus progesterone three times a week as previously described (36). The sham-operated and ovariectomized control rats were injected with corn oil (vehicle) in the same manner as the hormone-treated rats. The animal protocol was approved by the Experimental Animal Committee, Faculty of Science, Mahidol University, in accordance with the National Animal Laboratory Centre, Thailand.

SR Ca2+ uptake measurement. Ten weeks after surgery, the heart was rapidly removed under anesthesia and placed in ice-cold saline. The left ventricular homogenate was prepared and used for determining the oxalate-sustained SR 45Ca2+ uptake, as described by Pagani and Solaro (20) with modifications. Briefly, the whole left ventricle was homogenized in 20 mM imidazole with 30 passes in a Teflon-glass homogenizer, followed by filtration through six layers of cheesecloth. The left ventricular homogenate was then added to a final concentration of 1 mg/ml in the reaction mixture, containing 40 mM imidazole, 100 mM KCl, 4.5 mM MgCl2, 10 mM NaN3, 5 mM K+-oxalate, and 1 µM ruthenium red, at pH 7.0 and various concentrations of Ca2+ ranging from pCa 8.0 to 4.875 with 0.1% of 45CaCl2. The Ca2+ uptake reaction was started by the addition of ATP to a final concentration of 5 mM. The reaction mixture was then incubated for 3 min at 37°C with 60 rpm shaking and stopped by rapid cooling in ice. A portion of the reaction mixture was filtered through a 0.45-µm Millipore filter (Millex HA). An equal amount of filtrate and nonfiltrated solution was measured for radioactivity. The amount of Ca2+ uptake was calculated by subtracting the radioactivity of the filtrate from that of nonfiltrated radioactivity. Nonspecific binding was determined at pCa 5.0 in the absence of ATP. Protein concentration was measured with the Bradford assay.

SERCA activity. The SR membrane was prepared from the left ventricle by using the method of Jones et al. (19) with modifications. Immunoblot analysis revealed that the purification of the SR marker SERCA protein was 3.6 ± 0.5-fold in membrane preparations from the ventricular homogenate. This increase of purification is comparable to that previously reported (5). The SR-enriched membrane suspension was immediately frozen and stored at –80°C. Protein concentration of SR vesicles was determined by the Bradford assay. Activity of SERCA was determined by using triple enzyme assay as previously described by Chu et al. (7). In brief, the assay was run in various concentrations of Ca2+ ranging from pCa 8.0 to 5.0, pH 7.0 at 37°C. The SR-enriched membrane vesicles of 5 µg protein were added to the 1-ml reaction mixture, containing 21 mM MOPS, 4.9 mM NaN3, 0.06 mM EGTA, 100 mM KCl, 3 mM MgCl2, 0.2 mM NADH, 1 mM phospho(enol)pyruvate, 8.4 units of pyruvate kinase, and 12 U of lactate dehydrogenase. The ATPase reaction was started by the addition of ATP to a final concentration of 1 mM. SERCA activity was determined from the linear kinetic reaction of the NADH degradation monitored with a spectrophotometer at a wavelength of 340 nm. Nonspecific SERCA activity was analyzed in the mixture reaction of pCa 5.0 with the addition of 0.1 µM thapsigargin. Calculation of SERCA activity was analyzed from the optical density by using the extinction coefficient of NADH.

Semi-quantitative determination of SERCA mRNA by real-time PCR. Quantification of SERCA2a gene expression by real-time PCR was performed with the Rotor-Gene 3000 (Corbett, Sydney, Australia) using SYBR Green I nucleic acid stain (FMC BioProducts) and Rotor-Gene Analysis software version 5.0. Total RNA was isolated from the left ventricular tissue from the apex region of the hearts of each experimental group by using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instruction. Total RNA (2 µg) was reverse transcribed using SuperScript III First Strand Synthesis System (Invitrogen) with oligo-dT primer. PCR amplification was carried out in a 20-µl reaction mixture containing cDNA, 0.2 µM of each specific primer, 0.2 µM of each dNTP, 2.5 U of HotStartTag (Qiagen, Hilden, Germany), 0.5 mM MgCl2, 2 µl of 10x PCR buffer, and 1:40,000 SYBR Green I. PCR was conducted using denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s. The SercaF (5'-GACATTGAAACATGCTTTCTAATGGGC-3') and SercaR (5'-TGAACTGACGATGAGCACTTTATTACG-3') primers were synthesized according to Chu et al. (9). The ActinF (5'-CCTGGCACCCAGCACAAT-3') and ActinR (5'-GGGCCGGAC-TCGTCATAC-3') primers were used for amplification of beta-actin. SERCA mRNA levels were obtained after normalization with beta-actin mRNA.

Immunoblot analysis. The frozen left ventricular tissue was homogenized in the extracting buffer containing 150 mM Tris·HCl, 150 mM NaCl, 5 mM EGTA, 0.1% SDS, 50 mM NaF, 40 mM beta-glycerophosphate, 2 mM Na3VO4, and protease inhibitors, including leupeptin, pepstatin-A, aprotinin, and PMSF. Protein concentration of the left ventricular homogenate was determined by bicinchoninic acid assay. The homogenate with freshly added dithiothreitol was subjected to SDS-PAGE and immunoblot analysis. The protein content of SERCA and phospholamban in 100 µg of the left ventricular homogenate was quantified with monoclonal antibodies of SERCA2 (1:1,000) and phospholamban (1:5,000) (Affinity Bioreagents, Golden, CO). The proportion of the monomer to the pentamer of phospholamban was also quantified. Amounts of phosphorylated Ser16 (phospho-Ser16) and phospho-Thr17 phospholamban were determined in nondenatured preparations using polyclonal antibodies of phospho-Ser16 (1:20,000) and phospho-Thr17 phospholamban (1:5,000) (Badrilla, Leeds, UK). The relative amount of calsequestrin or actin was used for determining the total protein loading. Band density was analyzed by Image Master Labscan version 3.01 and Image Master Totallab version 1.0 (Amersham Pharmacia Biotech).

General methods and statistical analyses. Curves relating pCa and SR Ca2+ uptake or SERCA activity were fit to the Hill equation using nonlinear least squares regression analysis (GraphPad Prism, version 4.00) to derive the ECa50 (half-maximally activating calcium concentration) and the Hill coefficient (n). Data were presented as means ± SE. The significance of differences among groups of animals was analyzed using one-way ANOVA, followed by the Student-Newman-Keuls test for multiple comparisons. A P value of <0.05 was set for the significant difference among groups. The significance between the two groups was determined by a Student's t-test.

Materials. All chemicals were purchased from Sigma Chemical (St. Louis, MO). Some electrophoretic reagents were purchased from Bio-Rad (Hercules, CA) or Amersham Pharmacia Biotech (Buckinghamshire, UK). Thapsigargin was acquired from Alomone (Jerusalem, Israel), and radioactive 45CaCl2 was obtained from PerkinElmer (Boston, MA). Perioxidase-conjugated affinipure donkey anti-mouse IgG (H+L) was purchased from Research Diagnostics (Flanders, NJ), and horseradish peroxidase-goat anti-rabbit IgG (H+L) conjugate (ZyMax grade) was obtained from Zymed (San Francisco, CA).


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Deficiency of ovarian sex hormones in ovariectomized rats was verified by a significant decrease in the uterine weight compared with that of sham controls (Table 1). Restoration of uterine mass was clearly demonstrated in the estrogen-supplemented groups. Conversely, progesterone supplementation in ovariectomized rats could not maintain the uterine weight.


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  		Table 1. Body, LV, and uterine weights from Sham, OVX, OVX + E2, OVX + P, and OVX + E2P rats

 
The regulatory effects of ovarian sex hormones on the cardiac SR Ca2+ uptake were tested by oxalate-sustained SR 45Ca2+ uptake assay at various concentrations of calcium activation. Results depicted in Fig. 1 clearly show a significant suppression of the maximum SR Ca2+ uptake activity in ovariectomized hearts compared with those of sham controls. Surprisingly, a significant leftward shift in the pCa-Ca2+ uptake relationship was demonstrated in ovariectomized hearts with decreased EC50 values of 0.48 ± 0.02 µM compared with 0.59 ± 0.02 µM in sham controls (Fig. 2). Both the decreased maximum SR Ca2+ uptake and Ca2+ hypersensitivity of the cardiac SR Ca2+ uptake in ovariectomized rats was effectively prevented by supplementation with estrogen, progesterone, or the combined-hormone regimen. Furthermore, there was no change in the Hill coefficients of the pCa-Ca2+ uptake relations among the experimental groups (data not shown).


Figure 1
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  		Fig. 1. A: effects of ovariectomy on sarcoplasmic reticulum (SR) Ca2+ uptake of left ventricular homogenates at various calcium concentrations ranging from pCa 7.5 to 4.875, pH 7.0. B: comparison of maximum SR Ca2+ uptake between sham-operated control (Sham) and ovariectomized (OVX) rats with and without estrogen (E2) and/or progesterone (P) supplementation. Data are means ± SE from 8–9 preparations. *Significantly different from Sham (P < 0.05) using Student-Newman-Keuls test after ANOVA.

 

Figure 2
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  		Fig. 2. A: effect of ovariectomy on %maximum SR Ca2+ uptake of left ventricular homogenates at various calcium concentrations ranging from pCa 7.5 to 4.875, pH 7.0. B: comparison of EC50 between Sham and OVX rats with and without estrogen and/or progesterone supplementation. Data are means ± SE from 8–9 preparations. *Significantly different from Sham (P < 0.05) using Student-Newman-Keuls test after ANOVA.

 
To further elucidate the regulatory effect of ovarian sex hormones on SR Ca2+ uptake function, activity of the SERCA pump was determined at various Ca2+ concentrations using SR membrane preparations. In parallel to the SR Ca2+ uptake study, a significant decrease in the maximum SERCA activity (Fig. 3) and an increase in the Ca2+ responsiveness of SERCA activity (Fig. 4) were clearly detected in ovariectomized hearts. The magnitude of reduced EC50 in SERCA activity was found to be from 1.02 ± 0.12 µM in sham controls to 0.63 ± 0.06 µM in ovariectomized hearts. As expected, both the decreased maximum activity and the Ca2+ hypersensitivity of SERCA pumps in ovariectomized hearts were abolished in hormone-supplemented groups. There was also no difference in the Hill coefficients of pCa-SERCA activity relationship among the experimental groups.


Figure 3
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  		Fig. 3. A: pCa-SR Ca2+-ATPase (SERCA) activity relationship of SR membrane vesicles from Sham and OVX hearts. B: comparison of maximum SERCA activity between Sham and OVX rats with and without estrogen and/or progesterone supplementation. Data are means ± SE from 15–16 preparations. *Significantly different from Sham (P < 0.05) using Student-Newman-Keuls test after ANOVA.

 

Figure 4
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  		Fig. 4. A: effect of ovariectomy on %maximum SERCA activity at various calcium concentrations ranging from pCa 8.0 to 5.0, pH 7.0. B: comparison of EC50 between Sham and OVX rats with and without estrogen and/or progesterone supplementation. Data are means ± SE from 15–16 preparations. *Significantly different from Sham (P < 0.05) using Student-Newman-Keuls test after ANOVA.

 
We next determined the role of ovarian sex hormones in the regulation of the cardiac SR Ca2+ uptake activity. As shown in Fig. 5, a ~30% downregulation of SERCA proteins (Fig. 5, A and B), but with no change in the phospholamban content (Fig. 5C), was demonstrated in ovariectomized hearts compared with that of sham controls. The interactive ratio of SERCA to phospholamban protein content was significantly reduced in ovarian sex hormone-deficient hearts (Fig. 5D). Supplementation with either estrogen or progesterone completely abolished the downregulation of SERCA and restored the SERCA-to-phospholamban ratio in ovariectomized hearts. To further determine whether downregulation of SERCA was associated with its altered transcription level, quantification of the SERCA mRNA was performed using real-time PCR. As shown in Fig. 6, a ~70% reduction in the SERCA mRNA level was observed in ovariectomized hearts, with the level restored by estrogen or progesterone supplementation. The suppression of maximum SR Ca2+ uptake activity in ovarian sex hormone deficient-hearts was thus due to the decreased expression level of SERCA mRNA, and hence its protein, together with the greater inhibitory effect of phospholamban.


Figure 5
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  		Fig. 5. A: immunoblot analyses of SERCA, calsequestrin (CQ), and phospholamban (PLB) and comparisons of the band intensity expressed as a ratio of SERCA to CQ (B), PLB to CQ (C), and SERCA to PLB (D) of left ventricular homogenates from Sham and OVX rats with and without estrogen and/or progesterone supplementation. Data are means ± SE from 6–7 hearts. *Significantly different from Sham (P < 0.05) using Student-Newman-Keuls test after ANOVA.

 

Figure 6
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  		Fig. 6. Quantification of SERCA mRNA levels expressed as a ratio of SERCA to beta-actin of various ventricular homogenates from Sham and OVX rats with and without estrogen and/or progesterone supplementation. Data are means ± SE from 6 hearts. *Significantly different from Sham (P < 0.05) using Student-Newman-Keuls test after ANOVA.

 
To examine the modulating effect of phospholamban on the responsiveness of SERCA to Ca2+ in ovariectomized hearts, the content of the phosphorylated form of phospholamban at either the Ser16 or Thr17 site was analyzed. As illustrated in Fig. 7, there was no change in phospho-Ser16 phospholamban in ovariectomized hearts compared with the sham group. Unexpectedly, a significant reduction (~30%) in the amount of phospho-Thr17 phospholamban was clearly observed in ovariectomized hearts. The suppression of the phospho-Thr17 phospholamban in ovariectomized hearts was normalized by estrogen or progesterone supplementation (Fig. 8). These results indicated that changes in the SERCA response to Ca2+ after ovarian sex hormone deficiency were not modulated by alterations in phospholamban phosphorylation.


Figure 7
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  		Fig. 7. A: degree of phosphorylated Ser16 (phospho-Ser16) and phospho-Thr17 phospholamban and regions of actin on immunoblots of samples of left ventricular homogenates from Sham and OVX rats. B: relative amount of phosphorylated phospholamban to actin from each group. Data are means ± SE from 7–8 hearts. *Significantly different from Sham (P < 0.05) using a Student's t-test.

 

Figure 8
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  		Fig. 8. A: regions of phospho-Thr17 phospholamban and actin on immunoblots of samples of left ventricular homogenates from Sham and OVX rats with and without estrogen and/or progesterone supplementation. B: relative amount of phospho-Thr17 phospholamban to actin from each group. Data are means ± SE from 5–8 hearts. *Significantly different from Sham (P < 0.05) using Student-Newman-Keuls test after ANOVA.

 
On the basis of the suprainhibitory effect of monomeric phospholamban on the Ca2+ responsiveness of SERCA (8), the amount of the phospholamban monomer expressed as a percentage of the total phospholamban was determined in the nondenatured preparations of the left ventricular tissues. As shown in Fig. 9, a ~10% of the monomeric form of phospholamban detected in sham hearts was increased by about two folds in ovariectomized hearts. Such an increase was prevented by estrogen, progesterone, or the combination of estrogen and progesterone supplementation (Fig. 9C). These data confirmed that the Ca2+ hypersensitivity of SERCA activity induced by ovarian sex hormone deficiency was not underlied by changes in the amount or the activity of phospholamban.


Figure 9
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  		Fig. 9. A: regions of pentameric phospholamban and monomeric phospholamban on immunoblots of samples of left ventricular homogenates from Sham and OVX rats. B: amounts of phospholamban pentamer and monomer expressed as percentage of total phospholamban from each group. C: amount of monomeric phospholamban expressed as percentage of total phospholamban of samples of left ventricular homogenates from Sham and OVX rats with and without estrogen and/or progesterone supplementation. Data are means ± SE from 5–7 hearts. *Significantly different from Sham (P < 0.05) using Student-Newman-Keuls test after ANOVA.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our data are the first to provide evidence that ovarian sex hormones regulate SR Ca2+ uptake in the cardiomyocytes. This conclusion stems from the present finding that the depression in the maximum SR Ca2+ uptake activity observed in ovariectomized hearts resulted from the reduced expression levels of SERCA mRNA and protein and qualitative changes to the SERCA protein. The suppressed SERCA activity may have resulted in part from the pronounced inhibitory interaction of phospholamban on SERCA. Moreover, hormone supplementation studies demonstrated an equal contribution of estrogen and progesterone to the regulation of the SR Ca2+ uptake function. These alterations in SR Ca2+ uptake activity in ovariectomized hearts could be part of the overall changes in the intracellular Ca2+ handling, resulting in a lower magnitude and a prolonged decay of the Ca2+ transients previously reported (25).

Downregulation of SERCA in ovariectomized hearts is a result of a lower expression and a possibly increased degradation of the proteins. It is currently not known how ovarian sex hormones regulate the expression of SERCA in cardiac tissues. At the transcriptional level, it could be mediated by interaction of the ligand-bound receptors with hormone response elements on target genes. However, there was neither estrogen nor progesterone response elements found on the complete structural gene of the human SERCA and its 5'-regulatory region (41). In other instances, hormone-mediated transactivation could be induced without direct binding of the hormone receptor on DNA (11, 12, 21, 27, 29, 31, 33). In addition, the regulation of human SERCA gene expression in cardiomyocytes was found to be dependent on the transactivation events elicited by Sp1 and Sp3 transcription factors (2). Alternatively, the reduction of SERCA protein in ovariectomized hearts could involve a decrease in protein stability. In skeletal muscle, heat shock protein 70 (Hsp70) could bind to SERCA and therefore prevent thermal inactivation of the protein (32). Previous reports demonstrating a significant downregulation of Hsp70 in ovariectomized hearts (4) supported that the reduction in SERCA could be a result of increased protein degradation. Further studies on SERCA gene expression, including at the transcription initiation and posttranscriptional level, could give more insight into the mechanism of SERCA downregulation.

Another possible mechanism underlying regulation of SERCA activity by ovarian sex hormones is through modification of protein phosphorylation. In the heart muscle cells, the activity of SERCA was normally regulated by phosphorylation of SERCA via Ca2+/calmodulin-dependent protein kinase II (CaMK II) or by an integral SR phosphoprotein, phospholamban (13). Direct phosphorylation of SERCA by CaMK II at Ser38 increased the Vmax of Ca2+ pumping activity, serving to enhance the speed of cardiac muscle relaxation (30, 40). On the other hand, phosphorylation of phospholamban relieved its inhibitory effect on SERCA, leading to an enhanced affinity of the ATPase for Ca2+ and an increase in SERCA activity (15). Because PKA and CaMK II phosphorylate phospholamban on Ser16 and Thr17 residues, respectively (15, 37), a reduction in phospho-Thr17 phospholamban observed in ovariectomized hearts (Fig. 7) indicated a suppressed CaMK II activity and/or increased phosphatase activity in the cells. Because there was no change in the amount of phospho-Ser16 phospholamban but a reduction in the systolic Ca2+ concentration as reported earlier (25), a suppression of CaMK II activity was more likely induced. Thus a reduced maximum SERCA activity in ovariectomized hearts was probably due in part to a decreased SERCA phosphorylation.

Although the present results strongly implied a regulatory role of ovarian sex hormones in SR Ca2+ uptake activity through SERCA expression and modification, the absence of correlation between SERCA Ca2+ responsiveness and the regulatory effect of phospholamban in ovariectomized hearts suggested that other functional adaptations of SERCA might also be involved. Surprisingly, our results on analysis of SERCA protein (Figs. 5, 7, and 9) all opposed the enhanced SERCA Ca2+ sensitivity demonstrated in ovariectomized hearts. Previous findings of a higher SERCA Ca2+ sensitivity in male cardiomyocytes compared with that of female cardiomyocytes could support our results presented in this study (10). However, a higher level of phospho-Ser16 phospholamban, which could underlie the higher Ca2+ sensitivity of SERCA activity, was also demonstrated in male hearts. It is, therefore, not known how the increase in SERCA Ca2+ responsiveness was induced in ovariectomized hearts. Recently, a G protein-coupled receptor 30, bound by estrogen and uniquely localized in the endoplasmic reticulum where it rapidly initiated calcium mobilization, was reported (26). Whether this G protein-coupled receptor is present in the cardiac SR and could interact with estrogen to induce SR Ca2+ mobilization awaits further investigation.

The present study demonstrates that both quantitative and qualitative changes in SR Ca2+ uptake activity in ovariectomized hearts resembled those reported in the failing hearts. The decrease in SERCA activity from a reduction in SERCA protein levels and/or a greater inhibition of SERCA activity by phospholamban in end-stage heart failure has been suggested to impair the removal of cytosolic Ca2+, to decrease the SR Ca2+ load, and therefore to impair SR Ca2+ release, all of which were characteristics of the failing hearts (16). These similar findings in ovariectomized hearts and the failing hearts thus supported our hypothesis regarding the physiological significance of the ovarian sex hormones in the regulation of SR Ca2+ uptake activity.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported in part by Thailand Research Fund Grants RSA4480011 (to J. Wattanapermpool) and BRG4880005 (to J. Wattanapermpool), the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (Grant PHD/0121/2542 to T. Bupha-Intr and J. Wattanapermpool), and Mahidol University Grant (to J. Wattanapermpool).


   ACKNOWLEDGMENTS
 
We thank Drs. R John Solaro, Nateetip Krishnamra, and Pornpimol Rongnoparut for critical reading of the manuscript. We thank Dr. Prapon Wilairat for kind assistance with the measurement of SERCA activity, Dr. Arunee Tititunyanond for kindly providing beta-actin oligonucleotides and advice on real-time PCR, and Ariyaporn Thawornkaiwong for technical assistance on SERCA mRNA measurement.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Wattanapermpool, Dept. of Physiology, Faculty of Science, Mahidol Univ., Rama 6 Road, Bangkok 10400, Thailand (e-mail: tejwt{at}mahidol.ac.th)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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