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Am J Physiol Cell Physiol 292: C1625-C1635, 2007. First published December 13, 2006; doi:10.1152/ajpcell.00368.2006
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RECEPTORS AND SIGNAL TRANSDUCTION

Altered Ca2+ handling by ryanodine receptor and Na+-Ca2+ exchange in the heart from ovariectomized rats: role of protein kinase A

Gennadi M. Kravtsov ,* Kenneth W. L. Kam,* Jing Liu, Song Wu, and Tak Ming Wong

Department of Physiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China

Submitted 7 July 2006 ; accepted in final form 11 December 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Our previous study has demonstrated that ovariectomy (Ovx) significantly increased the left ventricular developed pressure (LVDP) and the maximal rate of developed pressure over time (±dP/dtmax) in the isolated perfused rat heart and the effects were reversed by female sex hormone replacement. In the present investigation, we studied the effects of Ovx for 6 wk on Ca2+ homeostasis that determines the contractile function. Particular emphasis was given to Ca2+ handling by ryanodine receptor (RyR) and Na+-Ca2+ exchange (NCX). 45Ca2+ fluxes via the RyR, NCX, and Ca2+-ATPase (SERCA) were compared with their expression in myocytes from Ovx rats with and without estrogen replacement. Furthermore, we correlated the handling of Ca2+ by these Ca2+ handling proteins with the overall Ca2+ homeostasis by determining the Ca2+ transients induced by electrical stimulation and caffeine, which reveals the dynamic changes of cytosolic Ca2+ concentration ([Ca2+]i) in the heart. In addition, we determined the expression and contribution of protein kinase A (PKA) to the regulation of the aforementioned Ca2+ handling proteins in Ovx rats. It was found that after Ovx there were 1) increased Ca2+ fluxes via RyR and NCX, which were reversed not only by estrogen replacement, but more importantly by blockade of PKA; 2) an increased expression of PKA; and 3) no increase in expression of NCX and SERCA. We suggest that hyperactivities of RyR and NCX are a result of upregulation of PKA. The increased release of Ca2+ through RyR and removal of Ca2+ by NCX are believed to be responsible for the greater contractility and faster relaxation after Ovx.

ovariectomy

THERE IS INCREASING EVIDENCE that estrogen regulates the cardiac function by direct action on the myocardium. Ovariectomy (Ovx) has been shown to affect the cardiac contractility (5, 22, 27, 28, 37). A recent study from our laboratory (15) has also shown that Ovx for 6 wk significantly increased the left ventricular developed pressure (LVDP) and maximal rate of developed pressure over time (±dP/dtmax) in the isolated perfused rat heart and the effects were reversed by female sex hormone replacement. Moreover, the amplitude of the electrically induced intracellular Ca2+ transient (E[Ca2+]i) was also enhanced in the cardiomyocytes following Ovx and the effect was reversed by female sex hormone replacement (5, 15). Together with a previous finding of increase in the Ca2+ responsiveness of myofilament activation in the heart of Ovx rats (37), these observations imply that estrogen may alter Ca2+ handling, thus altering the contractile function of the heart.

Like the arrival of an action potential, electrical stimulation depolarizes the sarcolemma, thus opening the voltage gated L-type Ca2+ channel, which allows influx of Ca2+ into the cardiomyocyte. Ca2+ entry triggers Ca2+ release from the sarcoplasmic reticulum (SR) via the ryanodine receptor (RyR) by the calcium-induced Ca2+ release mechanism (4, 7, 8, 33). The combination of Ca2+ influx via the L-type Ca2+ channel and its release from SR induces a marked increase in free intracellular Ca2+ ([Ca2+]i) level, allowing Ca2+ to bind to myofilament and turning on the contractile machinery (2, 32). After contraction, Ca2+ is removed from the cytoplasm as a result of reuptake of Ca2+ by the SR Ca2+-ATPase (SERCA) back to SR, extrusion of Ca2+ out of the cell by the Na+-Ca2+ exchange (NCX) and sarcolemmal Ca2+-ATPase, and uptake by mitochondrial Ca2+ uniporter to the mitochondrion (2, 6, 31). In the rat ventricular myocyte, SERCA and NCX have been shown to be responsible for removal of Ca2+ by >90% and ~7%, respectively, and are therefore the mechanisms mainly responsible for relaxation (1, 2, 13). It has been well documented that the expression and activity of L-type Ca2+ channels were increased following Ovx, which were reversed by replacement with estrogen (5, 14, 15, 22). On the other hand, Ovx failed to affect the expression of either Ca2+-ATPase (SERCA2a) or phospholamban (24). Other than a change in affinity to Ca2+ of RyR after Ovx (3) there was no study on Ovx on RyR. Nor was there any study on the effect of Ovx on NCX.

Very recently, we reported that the protein kinase A (PKA) activity was enhanced in the heart from Ovx rats and estrogen replacement restored the level to that of the control. Furthermore, we found that Ovx led to a significant increase in the L-type Ca2+ channel activity in cardiomyocytes and the effect was not only abolished by estrogen replacement, but more interestingly by blockade of PKA (15), suggesting that deficiency of estrogen after Ovx increases the PKA activity, which in turn increases the L-type Ca2+ channel activity. It is known that PKA regulates the open probability of RyR (21). There is also evidence that PKA is capable of activating NCX by means of phosphorylation of NCX complex proteins in the rat heart (25). It is likely that an enhanced PKA activity after Ovx may also alter Ca2+ handling by RyR and NCX in the heart.

In the present study, we first determined the Ca2+ fluxes across RyR and NCX, using direct measurement of 45Ca2+ and their expression. We also determined the Ca2+ fluxes across SERCA and its expression. Then we correlated the handling of Ca2+ by these Ca2+ handling proteins with the overall Ca2+ homeostasis by determining the Ca2+ transients induced by electrical stimulation and caffeine, which reveals the dynamic changes of [Ca2+]i in the heart. Besides, the PKA expression was determined. Results showed that 1) in the heart of Ovx rats, Ca2+ fluxes across RyR and NCX were significantly increased; 2) the effects of Ovx were reversed not only by estrogen replacement, but more importantly by blockade of PKA; and 3) expression of PKA in Ovx rats was increased.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental animals. Female Sprague-Dawley rats weighing 190–210 g were purchased from Charles River Breeding Laboratories (Wilmington, MA) and randomly divided in two groups. One group underwent bilateral Ovx while the other group underwent sham operation and served as a control. One week after Ovx, a subgroup of the rats (Ovx+E2) was subcutaneously implanted with a 60-day release pellet containing 1.5 mg of 17beta-estradiol (Innovative Research of America). The sustained-release pellets were designed to maintain estrogen concentration within the physiological range. All surgical procedures were performed under intraperitoneal anesthesia using pentobarbital sodium at a dose of 60 mg/kg.

The effects of Ovx were determined on the sixth week after removal of the ovaries. As we have shown in our previous study that 6 wk after Ovx, the contractile functions of the heart were markedly affected (15). Estrogen replacement lasted for 4 to 5 wk according to Kolodgie et al. (17), who showed that the effects of Ovx were reversed with estrogen replacement for 4 wk.

The protocol of this study was approved by the Committee on the Use of Experimental Animals for Teaching and Research, The University of Hong Kong.

Isolation of ventricular myocytes. Ventricular myocytes were isolated from the left ventricle from sham, Ovx, and Ovx+E2 Sprague-Dawley rats, using a collagenase perfusion method described previously (38). More than 80% of the cells were rod shaped and impermeable to Trypan blue. After isolation, they were allowed to stabilize for at least 30 min before experiments.

Preparation of SR and sarcolemmmal vesicles and measurement 45Ca2+ transports. Sarcolemmal and SR membranes were purified as described previously (23) either from a fresh isolated rat hearts or after perfusion for 30 min with the Krebs solution containing the inhibitors of PKA (0.5 µM PKI14-22 + 1 µM KT27582). The membrane-enriched fraction was stored at –70°C until use. Protein concentration was determined by the method of Lowry (20).

The ATP-dependent transport of 45Ca2+ in SR vesicles loaded with 5 mM K-oxalate was measured at room temperature (22°C) as described previously (23). The activity of SERCA for all groups of rats was defined as the difference between the rate of 45Ca2+ uptake in K-oxalate containing solution in the presence/absence of 10 µM cyclopiazonic acid, a specific inhibitor of SERCA (26).

The ryanodine-sensitive Ca2+ release from SR was estimated as ATP-dependent 45Ca2+ transport inhibited by ryanodine at room temperature (22°C) following a protocol described previously (23). The difference in uptake in the presence or absence of a specific blocker of RyR, 100 µM ryanodine, was defined as Ca2+ release via the RyR in all three groups of rats (23).

NCX activity was evaluated as specific Na+-dependent Ca2+ uptake following the protocol as described previously (23).

Immunofluorescence of PKA. Immunofluorescence labeling was conducted on ventricular myocytes from sham, Ovx, and Ovx+E2 rat. Mouse monoclonal antibodies [3B2] directed against PKA (Abcam, Cambridge, UK) were used. Enzymatically isolated rat cardiomyocytes were washed 3 times with PBS (pH 7.4) and fixed in freshly prepared 0.125% glutaraldehyde + 0.25% paraformaldehyde in PBS for 40 min (room temperature). Autofluorescence was reduced by treating the cells after fixation with 0.5% NaBH4 and 0.1% NaIO4 in PBS for 30 min (room temperature). Subsequently, cells were rinsed twice with PBS and permeabilized for 15 min with PBS containing 0.1% Triton X-100. After being washed in PBS, the cells were processed in the following manner: incubation in 5% porcine serum PBS (to blocked unreactive groups) for 30 min, followed by incubation in the primary antibody [3B2] at a dilution 1:200 in PBS containing 0.1% BSA (PBS-BSA buffer) for 30 h at 22°C. After rinses with PBS-BSA buffer, cells were exposed for 3 h (22°C) to fluorophore-conjugated secondary antibodies (Texas red rabbit polyclonal to mouse IgG H&L, Abcam) at dilution 1:800, washed with PBS and placed in the maintaining medium (50% glycerol in PBS). To determine nonspecific labeling, we also conducted control experiments without the primary antibody (data not shown). PKA immunoreactivity was determined with Olympus Fluoview Fv 300 confocal laser scanning microscope (Olympus Optical, Tokyo, Japan) equipped with a x40 oil-immersion objective lens. Images (1,024 x 1,024 pixels) were acquired in XY frame-scan mode. The fluorescence intensities of the cells were analyzed using Metamorph software (Universal Imaging, Downingtown, PA).

Western blot analysis for SERCA and NCX. For expression of SERCA2/NCX, 100 µg of SR vesicle/sarcolemmal membrane protein was blotted on one lane of 10% SDS-PAGE. Verification of equivalent total protein load was confirmed visually against actin band by Coomassie blue staining of the gels in parallel with actin. Proteins were transferred electrophoretically to polyvinylidene difluoride membranes (0.2 µm pore size; Bio-Rad) at 4°C in a transfer buffer containing 20% methanol with the Bio-Rad Trans-blot electrophoretic transfer system. After being blocked with Tris-buffered saline (TBS; composed of Tris, NaCl, and 0.1% Tween 20) containing 5% nonfat milk, the membranes were incubated overnight at 4°C with an anti-SERCA2 antibody at 1:1,000, anti-NCX at 1:200. The second antibody for SERCA protein determinations was anti-goat, while the secondary antibody for NCX protein was anti-mouse conjugated to horseradish peroxidase at 1:2,000 in 5% nonfat milk-TBS-Tween 20 for 1 h at room temperature. The proteins of SERCA2 and NCX were detected by the chemiluminescence method (ECL Western blotting detection; Amersham Biosciences). The film was scanned (Hewlett-Packard Scanjet XPA 7400C) and the intensity of the bands was calculated with image analysis software (Quantity One, version 4.2.2; Bio-Rad). To ensure equal amounts of protein were loaded on each lane, the membranes were stripped with stripping buffer and reblotted with a mouse anti-GAPDH antibody (1:6,000). The intensity of the protein bands was first normalized against the GAPDH and then normalized to value obtained for Sham, which was given an arbitrary value of 1.

Measurement of [Ca2+]i. A spectrofluorometric method with fura 2-AM as the Ca2+ indicator was used for measurement of [Ca2+]i in single ventricular myocytes (38). Loading of cells with fura 2-AM was performed as described previously (38). Fluorescent signals obtained at 340 nm (F340) and 380 nm (F380) excitation wavelengths at a sampling rate of 10Hz were recorded and stored in a computer for data processing and analysis. The F340/F380 ratio was used to represent changes in cytosolic calcium ([Ca2+]i) in the ventricular myocyte. Both the electrically (E[Ca2+]i) and caffeine-induced [Ca2+]i transients were measured as previously described (12, 23). The amplitudes of the E[Ca2+]i and of the caffeine induced [Ca2+]i transient represent the Ca2+ release from the SR and the Ca2+ content in SR and removal via the NCX. To measure the decay rate of the [Ca2+]i transients, we determined the {tau} value according to the equation [Ca]peak x e–t/{tau} + [Ca]baseline, where t represents the continuous variable of time. The decay rate of the E[Ca2+]i indicates mainly the reuptake of Ca2+ by SR Ca2+-ATPase and removal by sarcolemmal NCX (2, 19). The decay rate of the caffeine-induced [Ca2+]i transient reflects NCX activity because caffeine keeps the RyR open and therefore the decay rate of its transient represents Ca2+ efflux through the sarcolemmal NCX (2, 7, 30). The resting [Ca2+]i was measured with the electrical stimulation was off in some experiments.

To change the fluorescence signal into the concentration units, an internal calibration was performed on each cell. The maximum fluorescence ratio (Rmax) was determined by exposure of the cell to ionomycin (5 µM) in the presence of 1.25 mM Ca2+. Following estimation of Rmax, an excessive amount of Ca2+ chelator EGTA (5 mM) was added, which gives the minimum fluorescence ratio. The [Ca2+]i was calculated from experimental R340/380 with Kd of fura 2 for Ca2+ taken to be 224 nM (11).

Serum estrogen level. Rat serum was obtained from rats after decapitation followed by brief centrifugation. The E2 levels in the serum were measured using solid phase 125I radioimmunoassay technique (Diagnostic Research laboratory) according to the manufacturer's instruction. In addition to the serum estrogen level, the body weight was also monitored as a noninvasive physiological measure of estrogen depletion after Ovx.

Drugs and chemicals. Type-1 collagenase, fura 2-AM, caffeine, nitrendipine, ATP disodium salt, LaCl3, valinomycin, EGTA, NaN3, phenylmethylsulfonyl fluoride, pepstatin A, leupeptin, and cell-permeable PKA inhibitor fragment 14-22 (PKI14-22) were purchased from Sigma-Aldrich. PKA inhibitor (KT5720) was purchased from Tocris Cookson. Anti-SERAC2 and anti-NCX were purchased from AbCam. 45CaCl2 was purchased from Amersham Pharmacia. horseradish peroxidase-conjugated anti-goal and anti-mouse secondary antibodies were purchased from Santa Cruz. The ECL detection kit was from Amersham Pharmacia. All drugs were dissolved in deionized H2O or Krebs solution, except fura 2-AM and cyclopiazonic acid, which was dissolved in DMSO. The final concentration of DMSO was ≤0.01%, which itself had no effect on the heart.

Statistical analysis. Results are expressed as means ± SE. Comparisons between groups of data were made by one-way ANOVA, followed by the Newman-Keuls comparison test. A P level of <0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
General features of experimental animals. Six weeks after Ovx, the female rats exhibited a significant reduction in serum estrogen level accompanied by a significant increase in body weight and a significant reduction in the ratio of heart weight to body weight (Table 1). Estrogen replacement increased the serum estrogen to a level even higher than that of the female rats and restored the body weight and the ratio of heart weight to body weight to those of the sham operated female rats (Table 1).


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  		Table 1. General features of experimental animal

 
Effect of Ovx with or without estrogen replacement on activity of RyR. The release of 45Ca2+ via RyR from SR (Fig. 1) were significantly higher by 110% in the heart from Ovx rats than that from sham rats. Estrogen replacement restored the value to that of the sham rats.


Figure 1
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  		Fig. 1. Activity of ryanodine receptor (RyR) in the left ventricle from sham, ovariectomized (Ovx) and Ovx+E2 rats. The Ca2+ release via the RyR was defined as the difference in rate of 45Ca2+ uptake in the presence/absence of 100 µM ryanodine. Values are means ± SE of 4 independent experiments. *P < 0.05 vs. corresponding Ovx and Ovx+E2 groups.

 
Effect of Ovx with or without estrogen replacement on the activity and expression of NCX. The NCX activity exhibited a 306% increase in the ventricular myocytes from Ovx rats compared with those from the sham group (Fig. 2A). The effect was reversed by estrogen replacement. However, the protein expression of the exchanger was the same in the heart from all three groups of rats (Fig. 2B), indicating the increased NCX activity was not due to any change in its expression.


Figure 2
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  		Fig. 2. Activity and expression of Na+/Ca2+ exchanger (NCX) in the left ventricle from sham, Ovx, and Ovx+E2 rats. A: NCX activity was estimated as a specific Na+-dependent 45Ca2+ uptake. Values are means ± SE of 4 independent experiments. B: Western blot of NCX. Top, representative blots. Bottom, group results. The relative arbitrary unit for the sham group was assigned as 1. Data are expressed as fold increase over the sham group. Data are represented as means ± SE of 3 hearts for each group on the same blot. **P < 0.01 vs. corresponding Ovx and Ovx+E2 groups.

 
Effect of Ovx with or without estrogen replacement on the activity and expression of SERCA. As shown in Fig. 3A, there was no significant difference in SERCA activity in the heart from three groups of rats. Nor was there any difference in expression level of SERCA2 protein in the heart from different groups (Fig. 3B). The result indicates that neither the expression nor the activity of SERCA2 was changed by estrogenic status of the animal. The result indicates that the increased rate of removal of Ca2+ in the heart from Ovx rats was not due to any change in SERCA2 activity.


Figure 3
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  		Fig. 3. Activity and expression of SERCA in the left ventricle from sham, Ovx, and Ovx+E2 rats. A: SERCA activity was defined as the differences between the rates of 45Ca2+ uptake in K+-oxalate containing solution in the presence/absence of 10 µM cyclopiazonic acid. Values are means ± SE of 5 independent experiments. B: Western blot of SERCA2. Top, representative blots. Bottom, group results. The relative arbitrary unit for the sham group was assigned as 1. Data are expressed as fold increase over the sham group. Data are represented as means ± SE of 3 hearts for each group on the same blot.

 
Effect of Ovx with or without estrogen replacement on E[Ca2+]i transients in ventricular myocytes. The amplitude of E[Ca2+]i transient, which indicates the amount of Ca2+ release from SR upon excitation-contraction coupling, was significantly higher (see Fig. 4, A and B) in ventricular myocytes from Ovx rats than in those from sham rats. The decay rate ({tau} value) of the transient, which indicates the rate of removal of Ca2+ from the cytoplasm, was significantly smaller in ventricular myocytes from Ovx rats than that from sham rats (366.4 ± 22.2 vs. 519.8 ± 33.5 ms; n = 5, P < 0.05) (Fig. 4, A and C). Estrogen replacement restored the values to those of the sham operated female rats (Fig. 4, B–D). It should be noted, there was no significant difference in the resting cytosolic Ca2+ level among all three groups (Fig. 4D).


Figure 4
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  		Fig. 4. Electrically induced [Ca2+]i transient in the left ventricular myocytes from sham, Ovx, and Ovx + E2 rats. A: representative tracing from single left ventricular myocytes in response to 0.2-Hz electrical field stimulation with 15-ms pulse at 60 V. B: mean value of the amplitude. C: mean value of the decay rate constant ({tau}). D: resting [Ca2+]i level. N = 15–25 cells from 3–5 rats in each groups. Values are means ± SE. *P < 0.05 vs. corresponding sham and Ovx+E2 groups.

 
Effect of Ovx with or without estrogen replacement on caffeine-induced [Ca2+]i transient in ventricular myocytes. As shown in Fig. 5, A and B, the amplitude of the caffeine-induced [Ca2+]i transient, an indication of the Ca2+ content in SR, was >1.56 times higher in the ventricular myocytes from Ovx rats than that from sham rats (Fig. 5, A and B). Furthermore, the {tau} value was significantly smaller in the ventricular myocytes from Ovx rats than in that from sham rats (1,668 ± 150.2 vs. 2,670.3 ± 150.2 ms; n = 5; P < 0.05) (Fig. 5, A and C), indicating that the removal of Ca2+ by NCX is greater in the heart of Ovx rats. Estrogen replacement also restored the values to those of the sham rat (Fig. 5, A–C).


Figure 5
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  		Fig. 5. Caffeine-induced cytosolic Ca2+ concentration ([Ca2+]i) transients in the left ventricular myocytes from sham, Ovx, and Ovx+E2 rats. A: representative tracing from single left ventricular myocytes showing [Ca2+]i transients in response to 20 mM of caffeine. B: mean value of amplitude. C: mean value of the decay rate constant ({tau}). N = 20–25 cells from 3–5 rats in each groups. Values are means ± SE. *P < 0.05 vs. corresponding sham and Ovx+E2 groups.

 
Influence of PKA inhibitors on rynodine-sensitive 45Ca2+ transport and NCX activity. To determine whether the changes in RyR and NCX activities were due to an enhanced PKA activity, we measured the RyR and NCX activities in the heart of Ovx rats upon blockade of PKA with selective inhibitors (KT5720 and PKI14-22), known to inhibit both catalytic and regulatory subunits of PKA (10, 16). Treatment of isolated hearts from Ovx rats with 1 µM KT5720 plus 0.5 µM PKI14-22 for 30 min at room temperature significantly attenuated the enhancing effects of Ovx on 45Ca2+-release through RyR from SR (Fig. 6A) and NCX activity (Fig. 6B) in hearts from Ovx rats. It should be noted that both Ca2+ handling proteins of sham rats were not significantly affected by blockade of PKA (Fig. 6). The results indicate that the suppressed RyR and NCX activities in the heart from Ovx rats are due to enhanced PKA activity.


Figure 6
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  		Fig. 6. Effects of antagonists of protein kinase A (PKA; 1 µM KT5720 + 0.5 µM PKI14-22) on ryanodine-sensitive 45Ca2+ transport into sarcoplasmic reticulum (SR; A) and NCX activity (B) in the left ventricle from Sham and Ovx rats. SR and plasma membrane were isolated from hearts, which were perfused with the cocktail of PKA antagonists for 30 min at 22°C. Values are means ± SE of 6 independent experiments. *P < 0.05 vs. control and Ovx rats treated by inhibitors of PKA.

 
Expression of PKA in ventricular myocytes from normal and Ovx rats. To identify the PKA in situ, we used immunocytochemical techniques to localize this protein in rat ventricular myocytes from sham, Ovx, and Ovx+E2 rats. Confocal microscopy of adult cardiomyocytes revealed prominent staining for PKA in the Z-lines and also in other region of cells (Fig. 7A). These data are consistent with an immunofluorescent localization of the regulatory subunit of PKA in cardiac myocytes reported earlier (29). Analysis of fluorescence intensities of cardiomyocytes from different groups indicates that Ovx resulted in overexpession of PKA (the difference in intensity between Ovx and sham rats was 340%), and its effect was reversed by estrogen replacement (Fig. 7B).


Figure 7
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  		Fig. 7. Immunolocalization of PKA in cardiac myocytes from sham, Ovx, and Ovx+E2 rats. A: confocal images of ventricular myocytes from sham, Ovx, and Ovx+E2 rats stained with antibodies to PKA. A weak fluorescence of the myocytes, for which primary antibody was omitted, has been adjusted to zero background. B: fluorescence intensity of the whole myocytes isolated from sham, Ovx, and Ovs+E2 rats. Data represent the fluorescence intensity of 60 myocytes per animal. Four rats were used for each group. Values are means ± SE. *P < 0.001 vs. corresponding sham and Ovx+E2 groups.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The most novel and important observation of the present study is that Ovx for 6 wk led to marked increases in Ca2+ release from RyR and Ca2+ removal from NCX, and the effects were reversed by estrogen replacement. In contrast, Ovx did not change the Ca2+ re-uptake back to SR via SERCA. Thus, our results indicate that deficiency of estrogen due to Ovx increased the Ca2+ fluxes across three Ca2+ handling proteins, namely, L-type Ca2+ channel, as shown previously (15), and RyR and NCX as shown in the present study. Another equally important observation is that as in L-type Ca2+ channel (15), the increases in Ca2+ fluxes across RyR and NCX after Ovx were abolished by blockade of PKA, indicating that an enhanced PKA activity, which was shown to occur after Ovx for 6 wk (15), mediates the effects of Ovx on Ca2+ fluxes across these two Ca2+ handling proteins. Therefore, altered Ca2+ homeostasis in the heart after Ovx may be mainly due to an increased PKA activity, which in turn enhances the Ca2+ fluxes across the L-type Ca2+ channel, RyR, and NCX.

It has been shown that voltage-dependent mechanisms are involved in the regulation of SR Ca2+ content, which depends on the duration of the action potential (35). Two obvious voltage-dependent pathways for Ca2+ entry into cell are 1) via sarcolemmal L-type Ca2+ channels and 2) via reverse mode NCX. Moreover, it has been shown that in ventricular myocytes with overexpessing NCX there was a significant Ca2+ entry via the exchanger during the latter part of the Ca2+ transient (34). In a previous study (15), we demonstrated an increased L-type Ca2+ channel activity after Ovx. In the present study, we found an increased NCX activity. It is possible that the increased activities of the L-type Ca2+ channel and NCX could lead to an increased [Ca2+]SR in cardiomyocytes of Ovx. So we can suggest that Ovx may increase not only the release of Ca2+ from SR via RyR and removal of Ca2+ via the NCX the Ca2+ but also enhance the Ca2+ content in SR.

In the present study we found that 45Ca2+ efflux through the RyR was significantly increased after Ovx. In support of the finding, the amplitude of the E[Ca2+]i was also significantly increased after Ovx. The release of Ca2+ from SR via RyR is proportional to the concentration gradient across the SR membrane and can be predicted by following equation (36)

Formula
where [Ca2+]SR and [Ca2+]i are the free concentrations of Ca2+ inside SR and cytoplasm of myocytes, respectively, and k is the proportional constant, which can be used to estimate the extent of SR Ca2+ release per unit time. Since [Ca2+]i was found to be similar in cardiomyocytes from Ovx and sham rats (~185 nM), whereas the [Ca2+]SR was significantly higher in the heart of Ovx rats than that from sham rats (the amount of a free Ca2+ in SR was evaluated as a maximal increase [Ca2+]i after application of caffeine to cardiomyocytes), we would expect that the release of Ca2+ through RyR of the heart from Ovx rats should be greater by 1.6-fold than that from sham rats. Another important parameter, which can characterize the condition of RyR, is the steady-state Ca2+ leak. Direct measurement of the ryanodine-sensitive 45Ca2+ transport in SR showed that the activity of ryanodine channels was 2.1-fold higher. It is known that the open probability of RyR determines the Ca2+ release from the SR (18, 21). Recently, it has been shown that the type 2 RyR (RyR2)/Ca2+ release channel macromolecular complex in the heart includes PKA (21). PKA phosphorylation of RyR2 dissociates a 12,600-D polypeptide (FKBP12.6/calstabin2) from the channel resulting in increased open probability of RyR channels (18, 21). In our previous study we noted that the basal activity of PKA is increased in the heart from Ovx rats (15). In the present study we demonstrated that blockade of PKA with selective inhibitors, KT5720 and PKI14-22, markedly and significantly attenuated the enhanced release of Ca2+ via RyR, suggesting that increased PKA activity in Ovx rats may be mainly responsible for the increased Ca2+ release, presumably due to increased open probability of RyR induced by PKA.

The most interesting finding of the present study was that in the heart from Ovx rats, the NCX activity was markedly increased as shown by direct measurement of 45Ca2+ and a significantly shorter decay rate of the caffeine induced [Ca2+]i transient. We attempted to estimate and compare the relative contributions of SERCA and NCX to the removal of Ca2+ from the cardiomyocyte. The total Ca2+ flux ([Ca2+]total), which is responsible for Ca2+ removal, should represent the sum of following systems: [Ca]total = SERCA + NCX + sarcolemmal Ca2+-ATPase + mitochondrial Ca2+ uniporter (2). Since in our experiments the two last Ca2+-transporting systems were blocked by specific inhibitors (calmidazolium and Ru-360, respectively), we suggest contributions each of this system are <0.5% and these parameters can be neglected. We found that SERCA and NCX were 93.1% and 6.9% in the sham rats (Fig. 8), respectively, which is consistent with our previous observation in normal rats (23). Unexpectedly, this ratio drastically changed to 76.7% and 23.3% after Ovx (Fig. 8), indicating a marked increased contribution of NCX in removal of Ca2+. It should be cautioned that data on the Ca2+ removal from cytoplasm via SERCA and NCX were obtained from SR and plasma membrane preparations. To measure the integrated Ca2+ flux contributing to [Ca2+]i, the samples were isolated from the same ventricular myocytes of each rat simultaneously. Despite some differences in the purification SR and PM, the procedure to obtain SR and PM preparations was standardized. With these two preparations, we found that RyR and NCX activities in groups with and without estrogen were significantly different and that discrepancy in the activities SERCA or NCX between two rats belonging to the same group (for example, sham rats) does not exceed 5%. So, it does provide useful indication on the relative contributions of the two transporters. Unlike the RyR the increase in activity of NCX was not accompanied by an increase in expression in the heart from Ovx rats. The finding indicates that the increased NCX activity was not a result of its expression. It has recently been demonstrated that NCX1 of cardiomyocytes is dynamically phosphorylated by PKA in vitro (25), and the regulation of this phosphorylation is attributed to the existence of NCX1 macromolecular complex, which includes both the catalytic and regulatory subunits of PKA (29). Moreover, it has been shown that PKA-dependent phosphorylation of NCX1 increased NCX1 activity in both Xenopus oocytes expressing cardiac NCX1 and adult rat ventricular cardiomyocytes (25). In our previous study (15), we demonstrated an increased basal PKA activity in the heart from Ovx, suggesting that the increased NCX activity may result from an increased PKA activity in the heart from Ovx rats. In the present study, we found a marked inhibition of NCX activity after perfusion of isolated hearts from Ovx rats with blockade of PKA inhibitors, indicating that an increased PKA activity increases NCX activity in the heart from Ovx rats. A recent study (9) showed that in the intact rabbit ventricular myocyte, the NCX function was not altered by isoproterenol, which is well known to activate PKA. It should, however, be noted that in the present study we compared the NCX activity in the control and the Ovx rats in which the PKA activity was suppressed.


Figure 8
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  		Fig. 8. Relative contributions of SERCA and NCX to removal of Ca2+ from cytoplasm in left ventricular myocytes from sham (A), Ovx (B), and Ovx+E2 (C) rats. Left, time courses of the 45Ca2+ uptake by SERCA and NCX measured at 22°C in isolated SR and sarcolemmal membrane fractions, respectively. The total Ca2+ flux ([Ca]total) was the sum of Ca2+ uptake by SERCA and NCX. Right, comparison of the maximal activities of SERCA and NCX at steady state (~30 min) derived from time courses of Ca2+ uptake (left). The [Ca2+]total upon steady state was expressed as 100%. Values are means ± SE of 4 independent experiments.

 
Was the increased PKA activity due to an upregulation of the enzyme? Immunofluorescence studies in this study showed a significant increased PKA expression in cardiomyocytes from the Ovx rat, an effect reversed by estrogen replacement. The observation suggests that the increased PKA activity in cardiac myocytes from Ovx is due at least partly to its overexpression.

On the basis of the measurement of 45Ca2+, the SERCA2, which is mainly responsible for the removal of Ca2+ from the cytoplasm back into the SR, was not changed in terms of activity, consistent with the lack of expression of SERCA2 observed in the present study and a previous study (24). However, the total removal of Ca2+ by both SERCA and NCX, as calculated by 1/{tau}, was increased from 1.72/s in myocytes of the control to 2.98/s in electrically stimulated myocytes of the Ovx group. At the same time, the rate of Ca2+ removal from the NCX, as calculated from the time constant of the caffeine induced [Ca2+]i transient, was only increased from 0.34/s in control to 0.65/s in the Ovx group. The increase of 0.31/s in the rate of NCX dependent Ca2+ removal could not account for the significantly larger increase of 1.26/s in the rate of total Ca2+ removal. The observation suggests that SERCA activity may be enhanced in Ovx myocytes. It should be noted that the Ca2+ released from SR is much greater in response to caffeine than to electrical stimulation (9). The electrically induced Ca2+ transient is affected by many factors such as superficial Ca2+, potential sensitive Ca2+, Na+ channels, Na+/K+ pump of sarcolemma and surface potential, which can modify Ca2+ binding by protein and other molecules. These make the comparison of NCX based on the two transient difficult.

In conclusion, the results of present study indicates that 6 wk after Ovx, the activities of two key Ca2+-handling proteins, RyR and NCX, in cardiomyocytes were significantly enhanced, which was due at least partly to upregulation of PKA, which in turn increased its activity. The alterations of Ca2+ homeostasis after Ovx are believed to be responsible the increased contractility and faster relaxation as observed in our previous study (15).


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The study was supported by a grant from the Committee on Research and Conference Grants, The University of Hong Kong and a Cardiovascular Research Fund donated by ICST Holding, Hong Kong. K. W. L. Kam was supported by a Postgraduate Studentship from The University of Hong Kong and an Edward Youde Memorial Fund Scholarship.


   ACKNOWLEDGMENTS
 
We thank Dr. M. L. Fung for advice and Mr. C. P. Mok for assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. M. Wong, Dept. of Physiology, 4/F Laboratory Block, Faculty of Medicine Bldgs., 21 Sassoon Rd., Pokfulam, Hong Kong SAR, China (e-mail: wongtakm{at}hkucc.hku.hk)

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.

* G. M. Kravtsov and K. W. L. Kam contributed equally to this work. Back


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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