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Gender differences in sarcoplasmic reticulum calcium loading after isoproterenol

Laboratories of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, 27709; and Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710

Submitted 5 June 2003 ; accepted in final form 20 August 2003

	ABSTRACT

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Males exhibit enhanced myocardial ischemia-reperfusion injury versus females under hypercontractile conditions associated with increased sarcoplasmic reticulum (SR) Ca²⁺. We therefore examined whether there were gender differences in SR Ca²⁺. We used NMR Ca²⁺ indicator 1,2-bis(2-amino-5,6-difluorophenoxy)-ethane-N,N,N',N'-tetraacetic acid to measure SR Ca²⁺ in perfused rabbit hearts. Isoproterenol increased SR Ca²⁺ in males from a baseline of 1.13 ± 0.07 to 1.52 ± 0.24 mM (P < 0.05). Female hearts had basal SR Ca²⁺ that was not significantly different from males (1.04 ± 0.03 mM), and addition of isoproterenol to females resulted in a time-averaged SR Ca²⁺ (0.97 ± 0.07 mM) that was significantly less than in males. To confirm this difference, we measured caffeine-induced release of SR Ca²⁺ with fura-2 in isolated ventricular myocytes. Ca²⁺ release after caffeine in untreated male myocytes was 377 ± 41 nM and increased to 650 ± 55 nM in isoproterenol-treated myocytes (P < 0.05). Ca²⁺ release after caffeine addition in untreated females was 376 ± 27 nM and increased to 503 ± 49 nM with isoproterenol, significantly less than in male myocytes treated with isoproterenol (P < 0.05). Treatment of female myocytes with N^G-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase (NOS), resulted in higher SR Ca²⁺ release than that measured in females treated only with isoproterenol and was not significantly different from that measured in males with isoproterenol. Female myocytes also have significantly higher levels of neuronal NOS. This gender difference in SR Ca²⁺ handling may contribute to reduced ischemia-reperfusion injury observed in females.

neuronal nitric oxide synthase; ischemia

Several groups have reported gender differences in Ca²⁺ handling after isoproterenol (10, 25). Curl et al. (10) have reported that after treatment with isoproterenol, male rat myocytes have larger Ca²⁺ transients than females. Vizgirda et al. (25) showed that isoproterenol caused a significantly larger change in myocyte shortening in male than in female myocytes. However, neither of these studies examined gender differences in SR Ca²⁺ levels after isoproterenol. Together, our previous data and data in the literature suggest that there are gender differences in Ca²⁺ handling after isoproterenol that can lead to gender differences in ischemia-reperfusion injury. The goal of this study was to test the hypothesis that SR Ca²⁺ loading after isoproterenol was different in males and females and that this difference leads to gender differences in ischemia-reperfusion injury.

	MATERIALS AND METHODS

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Measurement of SR Ca²⁺ With 1,2-Bis(2-Amino-5,6-Difluorophenoxy)-Ethane-N,N,N',N'-Tetraacetic Acid

Langendorff perfusion. The NMR measurements of SR Ca²⁺ using 1,2-bis(2-amino-5,6-difluorophenoxy)-ethane-N,N,N',N'-tetraacetic acid (TF-BAPTA) were done in perfused rabbit heart because of enhanced TF-BAPTA loading into SR in rabbit compared with rats or mice. We find that 30% of TF-BAPTA loads into the SR of rabbit compared with 5% in rat. This enhanced SR loading of TF-BAPTA is likely related to differences in esterase activity. Because of the poor SR loading of TF-BAPTA in rodents, these studies can only be done in rabbit. Hearts from male and female Dutch Belted rabbits (1.2–1.6 kg) were isolated and perfused as described previously (4). Hearts were placed in a 30-mm NMR tube and maintained at 37°C by the perfusate buffer and the variable temperature unit of the NMR. Hearts were loaded with 1,000 ml of 5 µM TF-BAPTA as described previously (4). As with our previous studies, TF-BAPTA loading did not cause a reduction in left ventricular developed pressure (18). All animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 85-13, revised 1996).

19F-NMR measurements and calculation of SR Ca²⁺ concentration. 19F-NMR measurements were performed on a Varian 400-MHz wide-bore NMR spectrometer as described previously (4). We measured SR free Ca²⁺ concentration by calculating the chemical shift between the 6F resonance in TF-BAPTA, which is insensitive to Ca²⁺ binding and thus serves as a shift reference, and the 5F resonance, which shifts downfield on Ca²⁺ complexation. On the basis of the chemical shift difference, SR Ca²⁺ was calculated by using the equation described previously (5).

Measurement of SR Calcium Using Isolated Cardiac Myocytes

Adult rat cardiac myocytes were isolated as previously described (13). The freshly isolated myocytes were placed on laminin-coated plates and allowed to attach for 30 min before they were loaded with fura-2 AM. Cells on cover-slips in Hanks'-HEPES solution were placed on the stage of a microscope connected to a PTI spectrofluorometer. Cells were treated with or without 10 nM isoproterenol for 1 min. Another group was treated with 10 nM isoproterenol plus 1 µM N^G-nitro-L-arginine methyl ester (L-NAME), and another group was treated only with 1 µM L-NAME. The solution was changed to a 0 Na⁺-0 Ca²⁺ Hanks'-HEPES solution for 1 min (in the treated groups, the treatment was included in the 0 Na⁺-0 Ca²⁺ Hanks'), and then caffeine (20 mM) was added and the Ca²⁺ released was used as a measure of SR Ca²⁺ content. Previous studies have shown that, in contrast to rabbits, pacing rat myocytes results in a decreased or unchanged SR Ca²⁺ load compared with unstimulated resting conditions (16). We examined the effect of caffeine release in myocytes that were stimulated at 0.5 Hz versus unstimulated myocytes, and, consistent with previous studies, we found a slight decrease in SR Ca²⁺ after stimulation (data not shown). Since it was not necessary to pace rat myocytes to maintain SR Ca²⁺ levels, studies were performed on unstimulated myocytes.

Simulated Ischemia

To confirm that there were gender differences in susceptibility to ischemic injury in the adult rat myocytes used to measure SR Ca²⁺, we subjected myocytes to simulated ischemia by pelleting the myocytes as described previously (24). Male and female myocytes were treated with or without 10 nM isoproterenol for 1 min and then pelleted and covered with oil. Another group of myocytes was treated with 10 nM isoproterenol with or without L-NAME for 1 min and then pelleted and covered with oil. Cell viability was assessed with trypan blue as a function of time as described (24).

Western Blots

Myocytes were mixed with lysis buffer, homogenized, and snap frozen until Western blots were run. Proteins were separated by SDS-PAGE electrophoresis as previously described (9) and blotted to nitrocellulose membranes. Membranes were probed with the following primary antibodies: neuronal NO synthase (nNOS; Santa Cruz sc-648, 1:200); endothelial NO synthase (eNOS; Santa Cruz sc-654, 1:200); calsequestrin (Affinity Bioreagents PA1–913, 1:2,500); sarco-(endo)plasmic reticulum Ca²⁺-ATPase (SERCA; Affinity Bioreagents MA3–919, 1:1,000); and PLB (Affinity Bioreagents MA3–922, 1:500).

Statistics

Data are expressed as means ± SE. For comparison between two groups, a Student's t-test was used. For comparison of multiple groups, significance (P < 0.05) was determined by ANOVA, followed by a Fisher post hoc test.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
To test whether there were gender differences in SR Ca²⁺ loading under conditions of increased contractility, we used two methods to measure SR Ca²⁺ levels in males and females in the presence and absence of isoproterenol. We used the high dissociation constant (K_d) 19F-NMR Ca²⁺ indicator TF-BAPTA to measure the SR Ca²⁺ concentration in perfused rabbit hearts. TF-BAPTA loads into both the cytosol and SR in perfused rabbit hearts and can be used to measure the Ca²⁺ concentration in these compartments if the Ca²⁺ concentration is within the range of the K_d (5, 18). Under basal conditions, cytosolic Ca²⁺ is too low to be monitored accurately by TF-BAPTA. As described previously (4, 5), ionized SR Ca²⁺ can be measured from the shift difference between the Ca²⁺-insensitive 6F resonance and the Ca²⁺-sensitive 5F resonance of TF-BAPTA in the SR. As shown in Fig. 1, addition of isoproterenol results in an increase in SR Ca²⁺ in males from a baseline value of 1.13 ± 0.07 to 1.52 ± 0.24 mM (P < 0.05). Female hearts had a basal SR Ca²⁺ that was not significantly different from that observed in males (1.04 ± 0.03 mM), but addition of isoproterenol to females did not lead to a significant increase in SR Ca²⁺; SR Ca²⁺ was 0.97 ± 0.07 mM after addition of isoproterenol.

View larger version (19K): [in this window] [in a new window]   Fig. 1. Gender differences in sarcoplasmic reticulum (SR) Ca2+ after addition of 10 nM isoproterenol (Iso) measured by using 19F-NMR of 1,2-bis(2-amino-5,6-difluorophenoxy)-ethane-N,N,N',N'-tetraacetic acid (TF-BAPTA)-loaded rabbit hearts (n = 5). *Significantly different vs. males + isoproterenol.

TF-BAPTA has the advantage of directly monitoring SR Ca²⁺ in an intact, beating heart. However, with an SR Ca²⁺ concentration of 1 mM, TF-BAPTA is at the edge of the useful range for measuring Ca²⁺. We therefore wanted to use another method to confirm this gender difference in SR Ca²⁺ after isoproterenol addition. We used caffeine-induced release of SR Ca²⁺ measured with fura-2 in isolated ventricular rat myocytes (3, 12, 29). Typical fluorescent traces of caffeine-releasable Ca²⁺ are shown in Fig. 2. As shown in Fig. 2 and summarized in Fig. 3, the Ca²⁺ released after caffeine addition in untreated male myocytes was 377 ± 41 nM and increased to 650 ± 55 nM in isoproterenol-treated myocytes (P < 0.05). The Ca²⁺ release after caffeine addition in untreated females was 376 ± 27 nM and increased to 503 ± 49 nM with isoproterenol treatment. There was a significant difference in SR Ca²⁺ release between male and female myocytes treated with isoproterenol (P < 0.05).

View larger version (15K): [in this window] [in a new window]   Fig. 2. Typical fluorescent trace of SR Ca2+ release on caffeine addition to a male (A) and a female (B) myocyte loaded with fura-2. OD, optical density.

View larger version (28K): [in this window] [in a new window]   Fig. 3. Gender differences in SR Ca2+ after Iso estimated from caffeine-releasable Ca2+ (n = 5–18) in rat myocytes. Fluorescence was converted to Ca2+ by calibrating the minimum (Rmin) and maximum (Rmax) ratio by using ionomycin with excess EGTA and excess Ca2+. The dissociation constant (Kd) was taken as 224 nM. L-NAME, NG-nitro-L-arginine methyl ester. * Significantly different from gender-matched control (no Iso). #Significantly different from males + Iso.

Because we previously found that the protection in females was blocked by inhibition of NO synthase (NOS), we tested whether the male/female difference in SR Ca²⁺ was dependent on NOS. Since NOS is activated by Ca²⁺, we reasoned that the increased Ca²⁺ during adrenergic stimulation or other hypercontractile stimuli could have different effects, via differences in NO production, in males versus females. To test this hypothesis, we examined whether inhibition of NOS with L-NAME would reverse the attenuation of the isoproterenol-induced increase in SR Ca²⁺ observed in females. As shown in Fig. 3, treatment of female myocytes with isoproterenol and L-NAME resulted in an SR Ca²⁺ loading that was higher than that measured in females treated only with isoproterenol and was not significantly different from the SR Ca²⁺ measured in males treated with isoproterenol. L-NAME addition in the absence of isoproterenol had no significant effect on SR Ca²⁺ in either male or female myocytes.

We examined whether there were gender differences in SR Ca²⁺-handling proteins. As shown in Fig. 4A, male and female myocytes had similar levels of calsequestrin, SERCA, and PLB. We did find a striking gender difference in levels of nNOS (Fig. 4B). Female myocytes have significantly higher levels of nNOS than male myocytes. We found no significant difference in eNOS in male versus female myocytes. We also measured the rate of reuptake (or decay) of Ca²⁺ during the Ca²⁺ transient in males and females. As expected, the decay time of the Ca²⁺ transient was faster after isoproterenol in both males and females; however, there was no significant difference in the time to 50% decay of the Ca²⁺ transient in male versus female myocytes before or after addition of isoproterenol (Fig. 5).

View larger version (40K): [in this window] [in a new window]   Fig. 4. Western blots showing gender differences in protein levels. A: lack of gender differences in calsequestrin, SERCA, and phospholamban. Graphs show mean densitometry data in arbitrary units from 3 hearts; a standard was used to normalize data between gels. B: levels of neuronal (n) and endothelial (e) nitric oxide synthase (NOS) in extracts from male and female myocytes. Graphs show mean densitometry data in arbitrary units from 3 hearts; a standard was used to normalize data between gels. *Significantly different from females.

View larger version (20K): [in this window] [in a new window]   Fig. 5. Summary of data showing the decay time for the Ca2+ transient in male and female myocytes before and after Iso. Myocytes were paced at 0.5 Hz.

To determine whether there was a gender difference in ischemia-reperfusion injury in the rat myocytes used to measure SR Ca²⁺, male and female rat myocytes were treated with 10 nM isoproterenol for 1 min and then the myocytes were pelleted and covered with oil to simulate ischemia (24). As shown in Fig. 6A, after 4 h of simulated ischemia, male myocytes treated with isoproterenol had significantly more cell death than female myocytes with isoproterenol or male or female myocytes without isoproterenol. Consistent with the effects of L-NAME on SR Ca²⁺, Fig. 6B shows in a separate experiment that female myocytes treated with L-NAME just before isoproterenol had a significantly higher percentage of cell death than females treated with isoproterenol alone but a similar percentage of cell death to that of male myocytes treated with isoproterenol alone. Addition of L-NAME to male myocytes treated with isoproterenol did not alter the percentage of cell death.

View larger version (20K): [in this window] [in a new window]   Fig. 6. Male rat myocytes exhibit enhanced susceptibility to cell death. A: male and female myocytes treated for 1 min with and without 10 nM Iso and then subjected to 4 h of simulated ischemia. Shaded square, oxygenated control, males and females combined; , males with simulated ischemia; , females with simulated ischemia; , males with simulated ischemia + Iso; females with simulated ischemia + Iso. Cell viability was measured with trypan blue staining as described in MATERIALS AND METHODS; n = 5–10. * Significantly different from male simulated ischemia group. B: separate study in which we measured trypan blue staining in male and female myocytes after 4 h of O2 or 4 h of simulated ischemia (SI), SI + Iso, and SI + Iso + L-NAME. Before treatment (at t = 0), all myocytes had 20% trypan blue staining and there were no differences between groups. * Significantly different from gender-matched myocytes subjected to simulated ischemia.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The data in this study present the novel finding that females have lower SR Ca²⁺ than males after isoproterenol treatment. This reduced SR Ca²⁺ loading in females is mediated by NO, because addition of a NOS inhibitor restored the isoproterenol-induced SR Ca²⁺ level in females to that observed in males. We also present the new observation that female myocytes have significantly higher levels of nNOS than males. Together with the literature and our previous data, these data suggest that increased NO generation in females reduces the isoproterenol-induced increase in SR Ca²⁺. We further show that this decrease in SR Ca²⁺ in isoproterenol-treated females translates into reduced ischemia-reperfusion injury. This finding is consistent with numerous studies (11, 15, 17, 28, 30, 31) showing that decreased SR Ca²⁺ results in decreased ischemia-reperfusion injury. In further support of this hypothesis, we find that the reduced ischemia-reperfusion injury in isoproterenol-treated female myocytes is reversed with inhibition of NOS, which also restores SR Ca²⁺ to the level observed in males.

We used two complimentary methods to measure SR Ca²⁺ in males and females, before and after isoproterenol stimulation. Both methods revealed less SR Ca²⁺ load in females compared with males after treatment with isoproterenol. Because measurements of SR Ca²⁺ with TF-BAPTA are time-averaged during the cardiac cycle, these values will underestimate diastolic SR Ca²⁺. Furthermore, because addition of isoproterenol increases heart rate, it will decrease the percentage of the cardiac cycle spent in diastole, enhancing the underestimation of SR Ca²⁺ measured with TF-BAPTA after isoproterenol. This underestimation of SR Ca²⁺ likely explains why we see no increase in SR Ca²⁺ measured with TF-BAPTA after isoproterenol addition in females, whereas we do measure an increase in SR Ca²⁺ after isoproterenol with the use of caffeine release. However, we were interested in determining whether females exhibited less SR Ca²⁺ loading after isoproterenol than males, and both methods show a significantly lower SR Ca²⁺ in females versus males after isoproterenol. In further support of the hypothesis that NO mediates the gender difference in SR Ca²⁺ observed with isoproterenol, we find that inhibition of NOS with L-NAME enhances the isoproterenol-induced increase in SR Ca²⁺ in females, such that females treated with isoproterenol and L-NAME have an SR Ca²⁺ similar to that measured in males treated with isoproterenol. These data are consistent with the hypothesis that increased production of NO in females attenuates SR Ca²⁺ loading following adrenergic stimulation.

Data in the literature suggest several possible mechanisms by which NO could alter SR Ca²⁺ loading. Previous studies (22) have suggested that NO donors can modulate the isoproterenol-dependent phosphorylation of PLB. However, our previous studies (7) showed gender differences in ischemia-reperfusion injury in hearts from mice null for PLB, suggesting that the protection in females is not mediated by differences in PLB phosphorylation. Furthermore, we find no significant difference in the rate of Ca²⁺ reuptake in male versus female myocytes after addition of isoproterenol. NO could alter Ca²⁺ sensitivity of contractile proteins, or NO could directly alter SERCA or the ryanodine release channel (26, 27). NO is also reported to alter activity of the L-type Ca²⁺ channel (14), and such changes would be expected to alter SR Ca²⁺ loading.

We found significantly higher levels of nNOS in female versus male myocytes but no significant difference in levels of eNOS. This differs from our findings in extracts from whole hearts (9), where we found a slight, but significant, increase in eNOS in females. Whole heart extracts contain a mixture of myocytes and nonmyocytes; thus one cannot distinguish which cell type is responsible for the changes observed in whole heart extracts. The protein levels reported in this study were obtained from cardiomyocyte preparations, which were >95% cardiomyocytes.

There are conflicting data in the literature regarding the effects of nNOS on adrenergic stimulation. Barouch et al. (2) report that nNOS-knockout hearts have a decreased -adrenergic contractile response, whereas Ashley et al. (1) and Sears et al. (20) report that nNOS-knockout hearts have an increased -adrenergic contractile response and increased SR Ca²⁺. Our data would suggest that ablation of nNOS would have a more pronounced effect in females than in males, because male myocytes have little nNOS. Our data with female myocytes are consistent with the results of Ashley et al. (1) and Sears et al. (20), in that in females we find an increase in nNOS concomitant with attenuation of SR Ca²⁺ loading following adrenergic stimulation.

In summary, these data are the first to show that under conditions of hypercontractility, such as occur with isoproterenol treatment, females exhibit less SR Ca²⁺ loading than males. This attenuation of SR Ca²⁺ loading may be involved in the reduced ischemia-reperfusion injury observed in females under hypercontractile conditions. We further find that this gender difference in SR Ca²⁺ and ischemia-reperfusion injury is mediated by NO.

	FOOTNOTES

 

Address for reprint requests and other correspondence: E. Murphy, Laboratories of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709 (E-mail: murphy1{at}niehs.nih.gov).

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.

^* J. Chen and J. Petranka contributed equally to this work.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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