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(Circulation Research. 2006;98:403.)
© 2006 American Heart Association, Inc.

Integrative Physiology

Hypercontractile Female Hearts Exhibit Increased S-Nitrosylation of the L-Type Ca²⁺ Channel 1 Subunit and Reduced Ischemia/Reperfusion Injury

Junhui Sun, Eckard Picht, Kenneth S. Ginsburg, Donald M. Bers, Charles Steenbergen, Elizabeth Murphy

From the National Institute of Environmental Health Sciences (J.S., E.M.), Research Triangle Park, NC; Department of Physiology (E.P., K.S.G., D.M.B.), Stritch School of Medicine, Loyola University Chicago, Maywood, Ill; and Department of Pathology (C.S.), Duke University Medical Center, Durham, NC.

Correspondence to Dr Elizabeth Murphy, National Institute of Environmental Health Sciences, Laboratory of Signal Transduction, MD 2-03, Room F279, 111 TW Alexander Dr, Research Triangle Park, NC 27709. E-mail murphy1{at}niehs.nih.gov

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mechanisms underlying gender differences in cardiovascular disease are poorly understood. We found previously that, under hypercontractile conditions, female hearts exhibit significantly less ischemia/reperfusion injury than males. Here we show that male wild-type (WT) mouse hearts pretreated with 10 nmol/L isoproterenol before ischemia exhibited increased injury versus female hearts, but this relative protection in females was absent in eNOS^–/– and nNOS^–/– hearts. In isoproterenol-treated female versus male hearts, there was also more endothelial NO synthase (eNOS) associated with cardiomyocyte caveolin-3, and more neuronal NOS (nNOS) translocation to caveolin-3 during ischemia/reperfusion. S-nitrosothiol (SNO) formation was increased in isoproterenol-treated ischemic/reperfused hearts in all mouse genotypes, but only in WT mice was SNO content significantly higher in females than males. Using the biotin switch method, we identified the L-type Ca²⁺ channel 1 subunit as the predominant S-nitrosylated protein in membrane fractions, and following isoproterenol and ischemia/reperfusion male/female differences in SNO were seen only in WT hearts, but not in constitutive NOS^–/– genotypes. The isoproterenol-induced increase in L-type Ca²⁺ current (I_Ca) was smaller in females versus in males, but NOS blockade increased I_Ca in females. This gender difference in I_Ca in isoproterenol-treated myocytes (and abolition on NOS inhibition) was mirrored exactly in Ca²⁺ transients and SR Ca²⁺ contents. In conclusion, these data suggest that eNOS and nNOS both play roles in the gender differences observed in ischemia/reperfusion injury under adrenergic stimulation, and also demonstrate increased S-nitrosylation of the L-type Ca²⁺ channels in female cardiomyocytes.

Key Words: ischemia • gender • endothelial nitric oxide synthase • neuronal nitric oxide synthase • S-nitrosylation • L-type Ca²⁺ channel

	Introduction

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Nitric oxide (NO) plays an important role in modulating myocardial function in both health and disease.^1–4 A recent study suggested that NO regulates cardiac function by spatial confinement of NO synthase (NOS) isoforms, ie, endothelial NOS (eNOS) is localized in caveolae, where it regulates the L-type Ca²⁺ channel in the plasma membrane, and neuronal NOS (nNOS) is located in the sarcoplasmic reticulum (SR), where it regulates Ca²⁺ release from the SR.⁵ In addition to activating cGMP-dependent signaling pathways, NO can directly modify sulfhydryl residues of proteins through S-nitrosylation, which has emerged as an important posttranslational protein modification based on prototypic redox mechanisms in signal transduction.⁶ It has been shown that the cardiac SR Ca²⁺ release channel/ryanodine receptor (RyR2) can be poly-S-nitrosylated and activated by S-nitrosothiol (SNO) compounds in vitro.⁷ NO has also been reported to modulate the activity of SERCA2a^8,9 and the L-type Ca²⁺ channel,^10–12 but there is no direct evidence for S-nitrosylation of these Ca²⁺ transporters in situ.

Gender differences in cardioprotection have been reported,^13,14 and NO has been shown to play a role in mediating cardioprotection in females.^15,16 It has been shown previously that isoproterenol pretreatment increased ischemia/reperfusion injury to a greater extent in male hearts compared with females and that this protection in females was mediated by NO.¹⁵ Furthermore, the male/female differences in ischemia/reperfusion injury correlated with increased SR Ca²⁺ content in males following adrenergic stimulation.¹⁶ We hypothesize that the NO-dependent gender differences in SR Ca²⁺ loading following isoproterenol could be attributable to NO modulation of Ca²⁺ transporters in the SR or the sarcolemma,¹⁷ which would lead to a change in SR Ca²⁺ loading.^18–20 To test this hypothesis we examined whether hearts from female mice lacking eNOS (eNOS^–/–) or nNOS (nNOS^–/–) exhibited protection. We also used the biotin switch method²¹ to identify male/female differences in S-nitrosylation of proteins.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals
C57BL/6J mice, mice lacking eNOS, and mice lacking nNOS were obtained from The Jackson Laboratory (Bar Harbor, Maine). All animals were adult, reproductively viable, and between 12 and 15 weeks old at the time of experimentation. There were no differences in heart weight to body weight ratios between any of the genotypes or genders. All animals were treated in accordance with NIH guidelines and the Guiding Principles for Research Involving Animals and Human Beings.

Control, Isoproterenol, and NOS Inhibitor Treatment
Hearts from male (n=9, wild type [WT]; n=5, eNOS^–/–; n=5, nNOS^–/–) and female (n=9, WT; n=5, eNOS^–/–; n=6, nNOS^–/–) mice were Langendorff perfused²² and subjected to 30 minutes of perfusion, 20 minutes of no-flow ischemia, and 40 minutes of reperfusion. Another group of male (n=10, WT; n=5, eNOS^–/–; n=6, nNOS^–/–) and female (n=9, WT; n=5, eNOS^–/–; n=6, nNOS^–/–) hearts were pretreated with 10 nmol/L isoproterenol 1 minute before ischemia. A group of WT hearts from male (n=6) and female (n=6) mice were pretreated with 1 µmol/L N^G-nitro-L-arginine methyl ester (L-NAME) (a nonspecific NOS inhibitor) 5 minutes before ischemia, and a fourth group of WT hearts from male (n=6) and female (n=6) mice were pretreated with 1 µmol/L L-NAME 4 minutes before treatment with 10 nmol/L isoproterenol, beginning 1 minute before ischemia. In addition, a selective nNOS inhibitor, N⁵-(1-imino-3-butenyl)-ornithine (L-VNIO),²³ was also used (n=5 in each gender). All treatments were continued during 30 minutes of reperfusion, followed by 10 minutes of washout reperfusion. Recovery of left ventricular developed pressure (LVDP) was expressed as a percentage of preischemic LVDP, before drug administration. For infarct size, the protocol was the same except that reperfusion was extended to 2 hours. After that, the hearts were perfused with 1% 2,3,5-triphenyltetrazolium chloride (TTC) and incubated in TTC at 37°C for 15 minutes, followed by fixation in 10% formaldehyde. Infarct size expressed as the percentage of total area of cross-sectional slice through the ventricles.²⁴

Preparation of Membrane Fractions from Mouse Hearts
All preparative procedures were performed in the dark to prevent the light-induced cleavage of SNOs. The crude heart homogenate was obtained by homogenizing (3x5 s of Ultra Turrax T25 set at 22 000 rpm) a heart (0.10 to 0.16 g) on ice in 1.5 mL of buffer containing (in mmol/L) 300 sucrose, 20 imidazole-HCl (pH7.0), 1 EDTA, and 0.1 neocuproine. An EDTA-free protease inhibitor tablet (Roche Diagnostics Corp) was introduced just before use. Homogenates were centrifuged for 15 minutes at 3800g at 4°C, and the supernatant was filtered through cheesecloth and ultracentrifuged for 2 hours at 100 000g at 4°C. The pellet was resuspended on ice in 1.5 mL of buffer containing (in mmol/L) 600 KCl, 300 sucrose, 20 imidazole-HCl (pH7.0), 1 EDTA, 0.1 neocuproine, 0.025 leupeptin, and 0.25 Pefabloc using a dounce glass homogenizer. The resuspended pellets were incubated on ice for 1 hour, followed by a 100 000g ultracentrifugation at 4°C. The membrane fraction was obtained by resuspension of the pellet in 0.5 mL of homogenization buffer.

SNO Determination
NO decomposition from SNOs in WT heart (n=3 in each gender) membrane fractions was initially detected with an Apollo 4000 Free Radical Analyzer equipped with an NO-selective sensor (WPI, Sarasota, Fla) using Cu²⁺ as a catalyst to release NO. Because this method required a large amount of samples, we later used a 2,3-diaminonaphthalene (DAN) fluorometric method for SNO determination in the remaining WT hearts and all NOS^–/– hearts.²⁵

Detection of S-Nitrosylation by Biotin Switch Method
S-Nitrosylated proteins were detected by a modification of the biotin switch method.²¹ Free thiols in heart membrane fractions were blocked by addition of 20 mmol/L methyl methanethiosulfonate to the homogenate buffer in the presence of 1% (wt/wt) SDS for 20 minutes at 50°C. Then free methyl methanethiosulfonate was removed by protein precipitation with addition of prechilled acetone and the mixture was incubated at –20°C for 20 minutes. After centrifugation at 3000g for 15 minutes at 4°C, the pellet was resuspended in 100 µL of homogenization buffer per milligram of protein in the starting sample, and 1 mmol/L sodium ascorbate was added to reduce SNO groups to free thiols which can react with 1 mmol/L of N-[6-(biotinamido)hexyl]-3'-(2'-pyridyldithio)propionamide (Biotin-HPDP, Pierce). After incubation for 1 hour at 25°C in the dark to biotinylate proteins that were originally S-nitrosylated, the mixture was divided into 2 aliquots: 1 for anti-biotin western blot, the other for immunoprecipitation (IP) with anti-DHPR1. The precipitants were immunoblotted with anti-DHPR1 and anti-biotin antibody, respectively. Equal amounts of protein were loaded on to all gels. Gel electrophoresis and Western blotting were performed under nonreducing conditions, followed by enhanced chemiluminescence detection.

Immunoprecipitation
For caveolin-3 IP, 200 µg of membrane fractions were incubated overnight in IP buffer containing monoclonal anti–caveolin-3 antibody, the aliquots of precipitated extracts were probed with anti–caveolin-3, anti-eNOS, and nNOS antibodies, respectively. For DHPR1 IP, after the biotin switch assay, 100 µg of S-nitrosylated/biotinylated membrane fractions were incubated with goat polyclonal anti-DHPR1 antibody, and precipitates were probed with anti-biotin and anti-DHPR1 antibodies, respectively.

Intracellular Ca²⁺ and I_Ca Measurements
For intracellular Ca²⁺ measurement, cardiomyocytes were placed on laminin-coated glass coverslips and allowed to attach for 30 minutes before they were loaded with fura-2/acetoxymethyl ester (Molecular Probes). Measurements were made on a Photon Technology International spectrofluorometer. After measurement of basal Ca²⁺ concentration, myocytes were field stimulated (20 V) at 0.5 Hz for 1 minute, followed by 1 minute of treatment with 10 nmol/L isoproterenol. L-NAME (1 µmol/L) was added during the last 5 minutes of fura-2 loading and present throughout the experiment. Field stimulation was stopped 20 s before caffeine (20 mmol/L) addition and caffeine releasable Ca²⁺ was used as a measure of SR Ca²⁺ content.

To measure Ca²⁺ influx via sarcolemmal Ca²⁺ channels, cardiomyocytes were loaded with fluo-4/acetoxymethyl ester for 20 minutes and pretreated with 0.5 µmol/L thapsigargin for 15 minutes to completely block SR function, which was verified by the abolition of caffeine-induced Ca²⁺ transients. To enhance the small Ca²⁺ influx-dependent Ca²⁺ transients, superfusate [Ca²⁺] was raised to 20 mmol/L. Under these conditions, modest amplitude Ca²⁺ transients were measured which are attributable mainly to Ca²⁺ influx. Treatment with isoproterenol and L-NAME was as described above.

For I_Ca measurements, experiments were performed using rat cardiomyocytes²⁶ under voltage clamp using the perforated patch technique with ß-escin. The pipette solution contained (in mmol/L) 125 CsCl, 20 TEA-Cl, 10 HEPES, 5 phosphocreatine, 5 Mg₂ATP, 0.04 ß-escin; titrated to pH 7.2 with CsOH. The extracellular solution contained (in mmol/L) 140 NaCl, 4 CsCl, 1 MgCl₂, 1 CaCl₂, and 10 HEPES, pH 7.4. Membrane potential was held at –80 mV and I_Ca was measured by applying a 50-ms prepulse to –50 mV and a 200-ms test pulse to 0 mV at 0.33 Hz. Cells were stimulated at least 20 times before isoproterenol (100 nmol/L) was added. Increase in I_Ca peak current was normalized to the peak current without isoproterenol. Treatment with L-NAME was as described above.

Data Analysis
Results are expressed as means±SE. Statistical significance was determined by Student’s t test or ANOVA as required. Differences were regarded to be significant at P<0.05.

	Results

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Recovery of LVDP After Ischemia/Reperfusion
Consistent with our previous study in 129J mice,¹⁵ male WT C57BL/6J mice exhibited enhanced injury compared with females when the hearts were pretreated with 10 nmol/L isoproterenol for 1 minute before ischemia. Postischemic recovery of LVDP was 13±2% in male hearts compared with 31±2% in females (Table). The male/female differences were attenuated significantly if 1 µmol/L L-VNIO, a selective nNOS inhibitor, was introduced before perfusion with 10 nmol/L isoproterenol, whereas 1 µmol/L L-NAME, a nonspecific NOS inhibitor, completely blocked the male/female differences (Figure 1A). As shown in Figure 1B, there were no male/female differences in infarct size after 20 minutes of ischemia in the absence of isoproterenol; however, with isoproterenol treatment, males exhibited more necrosis than females. This male/female difference in infarct size could be partially blocked or totally abolished by the pretreatment with L-VNIO or L-NAME, respectively. Thus, the gender differences in ischemia/reperfusion injury under adrenergic stimulation were mediated through a NOS dependent mechanism. In addition, the reduction (by L-VNIO) or abolishment (by L-NAME) of the gender differences suggests that both constitutive NOS isoforms, eNOS and nNOS, may contribute to this gender difference.

View this table: [in this window] [in a new window]   Table 1. LVDP Recovery in Ischemic-Reperfused Mouse Hearts and S-Nitrosothiol Content in Heart Membrane Fractions

View larger version (37K): [in this window] [in a new window]   Figure 1. Postischemic recovery of LVDP (A) (n=9 to 10 in each control and +isoproterenol [Iso]-treated group; n=4 to 5 in each +Iso+NOS inhibitor group) and infarct size (B) (n=4 to 5 in each group) following adrenergic stimulation in WT C57BL/6J mouse hearts. *P<0.05 vs male with same treatment, #P<0.05 vs female+Iso.

To further determine the role of the constitutive isoforms of NOS in the male/female differences in ischemia/reperfusion injury following adrenergic stimulation, we investigated eNOS^–/– and nNOS^–/– mice. As shown in the Table, in the absence of isoproterenol, postischemic recovery of LVDP was similar in WT and nNOS^–/– hearts, whereas eNOS^–/– hearts exhibited significantly poorer recovery of function. Thus in the absence of isoproterenol eNOS may improve postischemic recovery in both male and female mice.

After isoproterenol treatment in eNOS^–/– hearts, postischemic recovery of LVDP was 12±2% in male and 15±3% in female hearts. Thus, the male/female differences observed in WT hearts were not observed in hearts from mice lacking eNOS. Similarly, in nNOS^–/– hearts treated with isoproterenol before ischemia, postischemic recovery of LVDP was comparably poor in males versus females (15±3 versus 16±3%). These data suggest that the presence of both eNOS and nNOS is necessary for the NOS-dependent protection observed in females after isoproterenol. The inotropy induced by isoproterenol is associated with an increase in Ca²⁺, which could activate both eNOS and nNOS, providing an explanation for the observation that the male/female differences are only discernable on adrenergic stimulation.

SNO Contents in Heart Membrane Fractions
In nonischemic WT mice, male and female hearts contained nearly equal amounts of SNOs, ie, 4 pmol/mg protein in heart membrane fractions (Table). Interestingly, the SNO signal was barely detectable in eNOS^–/– heart membrane fractions, whereas nNOS^–/– hearts had basal levels of SNO similar to those observed in WT. The SNO content in heart membrane fractions was not significantly altered by ischemia/reperfusion. However, hearts treated with isoproterenol before ischemia/reperfusion showed a dramatic increase in SNO content, with the largest amount of SNO in WT female hearts, followed by male WT, then nNOS^–/– and finally eNOS^–/– hearts. Consistent with the observation that only female WT hearts were protected, we observed a significant male/female difference in SNO only in WT hearts but not in eNOS^–/– or nNOS^–/– hearts. A recent study by Ghelardoni et al did not find any detectable SNO in crude membrane fractions of perfused rat heart after ischemia or postischemic reperfusion, but they did detect SNO formation after perfusion with NO donors.²⁵ In the work described herein, addition of EDTA and neocuproine (a specific copper chelator) to the extraction buffers and preparative procedures in the absence of light enhanced our sensitivity.

S-Nitrosylation of L-type Ca²⁺ Channel/DHPR1 Subunit
To identify potential targets for the NO induced protection observed in females, a modified biotin switch method²¹ was used to identify S-nitrosylated proteins in hearts. After specific biotinylation of endogenously S-nitrosylated cysteine residue(s), an anti-biotin immunoblot was used to identify these biotinylated proteins. Surprisingly, only a few bands were observed in the anti-biotin immunoblots; the most predominant band had a molecular mass of 220 kDa (Figure 2A). The presence of this band in nonischemic hearts suggests that this protein is endogenously S-nitrosylated. The molecular weight of this band is consistent with the L-type Ca²⁺ channel/dihydropyridine receptor (DHPR) 1 subunit. To confirm that this biotin labeled protein was the DHPR1, we stripped and reprobed with anti-DHPR1 antibody, and, as shown in Figure 2B, immunoreactive bands were observed at the same position (220 kDa). We then performed a number of studies summarized in Figure 2C. The level of S-nitrosylation of this protein was not increased in standard ischemic/reperfused hearts, whereas an increase was found in nonischemic hearts with isoproterenol pretreatment. Moreover, with isoproterenol the increase in S-nitrosylation was greater in female than male hearts. The S-nitrosylation signal was significantly augmented in ischemic/reperfused hearts with isoproterenol pretreatment, and females had a significantly higher S-nitrosylation level than males. However, in the presence of NOS inhibitors, the S-nitrosylation of this protein was significantly attenuated, and there were no significant differences between males and females (Figure 2C).

View larger version (27K): [in this window] [in a new window]   Figure 2. S-Nitrosylated protein(s) identified by the biotin switch method. A, Anti-biotin immunoblot following the biotinylation of S-nitrosylated protein(s). B, Anti-DHPR1 immunoblot after the membrane for anti-biotin detection (left) was stripped and reprobed. Lanes 1 and 5, Nonischemic (Non-Isch) heart without Biotin-HPDP treatment as negative control. Lanes 2 and 6, Nonischemic heart as background control. Lanes 3 and 7, Ischemic/reperfused (Isch-Rep) heart. Lanes 4 and 8, Ischemic/reperfused heart with 10 nmol/L isoproterenol (Iso) pretreatment. C, Mean values for the S-nitrosylation level of 220-kDa protein. The densitometry data were normalized (n=3 to 5) to the anti-biotin signal of nonischemic male hearts. *P<0.05 vs nonischemic hearts in each gender, #P<0.05 vs male with same treatment, P<0.05 vs standard ischemic/reperfusion in each gender.

To further confirm that the predominant S-nitrosylated/biotinylated protein is the DHPR1 and to examine effects in eNOS^–/– and nNOS^–/– mice, we immunoprecipitated the biotinylated membrane fractions with anti-DHPR1 antibody and probed with anti-DHPR1 and anti-biotin antibody, respectively (Figure 3). Interestingly, the baseline level of S-nitrosylation of DHPR1 appeared to increase with the order of eNOS^–/–<nNOS^–/–<WT. Ischemia/reperfusion with isoproterenol pretreatment increased S-nitrosylation levels in WT, eNOS^–/–, and nNOS^–/– hearts; however, only in the WT group did female hearts have a significantly higher level of S-nitrosylation in DHPR1 than male hearts. Consistent with the lack of male/female differences in postischemic function, eNOS^–/– and nNOS^–/– hearts did not show a significant gender differences in S-nitrosylation in DHPR1 following ischemia/reperfusion, and female eNOS^–/– and nNOS^–/– hearts were similar to male WT.

View larger version (25K): [in this window] [in a new window]   Figure 3. S-Nitrosylation of L-type Ca2+ channel/DHPR1 subunit. A, The biotinylated/S-nitrosylated proteins in heart membrane fractions were immunoprecipitated with polyclonal anti-DHPR1 antibody, and the precipitants were detected by anti-DHPR1 and anti-biotin immunoblot (IB), respectively. Odd lanes, Nonischemic (Non-Isch) hearts. Even lanes, Ischemic/reperfused (Isch-Rep) hearts with 10 nmol/L isoproterenol (Iso) pretreatment. B, Mean densitometry data from 3 individual experiments were normalized to the anti-biotin signal of nonischemic eNOS–/– male hearts. *P<0.05, significantly different vs nonischemic hearts in each gender of individual genotype of mice; #P<0.05, significantly different vs ischemic/reperfused WT male hearts with isoproterenol pretreatment; P<0.05, significantly different vs ischemic/reperfused WT female hearts with isoproterenol pretreatment.

Expression and Distribution of eNOS and nNOS in Mouse Hearts
eNOS has been shown to be localized to caveolae in the plasma membrane,^5,27,28 where eNOS would be in close proximity with some L-type Ca²⁺ channels and on activation eNOS would result in S-nitrosylation of L-type Ca²⁺ channels.²⁹ The increase in S-nitrosylation of the L-type Ca²⁺ channel in WT females (see Figures 2 and 3) would suggest an increase in eNOS levels in females. To examine this hypothesis, we immunoprecipitated isolated membrane fractions with anti–caveolin-3, the cardiomyocyte specific caveolin.²⁷ WT females showed an increase in eNOS association with caveolin-3 under all conditions (Figure 4). In contrast to eNOS, nNOS has been suggested to localize to the SR,^5,8 although recent data suggest that nNOS can translocate to the sarcolemma following myocardial infarction³⁰ or in failing hearts.³¹ Furthermore, Sears et al have reported that nNOS can modulate L-type Ca²⁺ channels in the plasma membrane.³² As shown in Figure 4, at baseline (nonischemic hearts) we see little or no association of caveolin-3 and nNOS. However, with ischemia and reperfusion following isoproterenol, there is a significant increase in association between nNOS and caveolin-3, and this association is significantly higher in females versus males. Thus both eNOS and nNOS are in a location where they can contribute to the increase in S-nitrosylation of the L-type Ca²⁺ channel in WT female cardiomyocytes exposed to isoproterenol and ischemia/reperfusion.

View larger version (41K): [in this window] [in a new window]   Figure 4. Expression and distribution of eNOS and nNOS in WT mouse hearts. A, Following caveolin-3 IP, the immunoprecipitant (bound beads, lanes 1 to 4) and supernatant (unbound, lanes 5 to 8) were probed with anti–caveolin-3, anti-eNOS, and anti-nNOS antibodies. Odd lanes, Nonischemic (non-isch) hearts. Even lanes, Ischemic/reperfused (Isch-Rep) hearts with 10 nmol/L isoproterenol (Iso) pretreatment. B, Densitometry data of anti-eNOS and anti-nNOS from 3 to 4 individual immunoprecipitant experiments were normalized with male nonischemic hearts. *P<0.05, significantly different from male nonischemic hearts; #P<0.05, significantly different from male ischemic/reperfused hearts with 10 nmol/L isoproterenol.

Male/Female Differences in Ca²⁺ Transient and I_Ca
As shown in Figure 5, there was no gender difference in cytosolic free Ca²⁺ in unstimulated mouse cardiomyocytes, which was 92.5±18.1 nmol/L in males and 84.8±12.7 nmol/L in females. Perfusion with 10 nmol/L isoproterenol significantly increased peak Ca²⁺ amplitude in both male and female cardiomyocytes to 335.4±58.3 and 251.6±36.5 nmol/L, respectively. The peak systolic Ca²⁺ following isoproterenol stimulation was lower in females compared with that in males, which has been also reported in rat cardiomyocytes.³³ Consistent with our previous finding in rat cardiomyocytes,¹⁶ SR Ca²⁺ content measured using caffeine-induced Ca²⁺ release was significantly lower in females than in males, 510.3±96.3 versus 751.6±105.4 nmol/L. The peak Ca²⁺ amplitude after isoproterenol or caffeine-induced Ca²⁺ release normalized to the peak systolic Ca²⁺ before addition of isoproterenol were both significantly lower in females than in males. These results show a male/female differences in Ca²⁺ transient and SR Ca²⁺ content in cardiomyocytes under adrenergic stimulation; females treated with isoproterenol have a lower systolic Ca²⁺ and less of an increase in SR Ca²⁺. Moreover, this male/female difference is abolished by L-NAME.

View larger version (30K): [in this window] [in a new window]   Figure 5. Intracellular Ca2+ measurement in cardiomyocyte with intact SR function. A and B, Representative traces of fura-2 fluorescence (340/380) in single cardiomyocytes isolated from male (A) and female (B) mouse hearts. Fluorescence was recorded before and following field stimulated pacing, 10 nmol/L isoproterenol and 20 mmol/L caffeine, which were added as indicated. C, Ratio of peak Ca2+ amplitude after isoproterenol or caffeine-induced Ca2+ release normalized to the peak amplitude before addition of isoproterenol. L-NAME (1 µmol/L) was added into buffer as described in Materials and Methods to prevent S-nitrosylation. Compared with male, *P<0.05. The number of cells and animals was indicated in the parenthesis.

To further assess Ca²⁺ influx, Ca²⁺ transients ([Ca]_i) in cardiomyocytes were recorded with SR function blocked by pretreatment with 0.5 µmol/L thapsigargin (Figure 6). The lack of SR function is reflected in the small Ca²⁺ transient size, the very slow [Ca]_i decline and insensitivity of [Ca]_i decline to isoproterenol. Perfusion with 10 nmol/L isoproterenol increased Ca²⁺ transient amplitude in males to 140±10% of control (n=18) but in females only to 115±7% of control (n=20; P<0.05). Pretreatment with L-NAME had no effect on the isoproterenol-induced Ca²⁺ transient amplitude in males (142±11%, n=16) but enhanced the increase in Ca²⁺ transients in the female mice (144±12%, n=12). That is, the gender difference in isoproterenol response was abolished by L-NAME.

View larger version (48K): [in this window] [in a new window]   Figure 6. Intracellular Ca2+ measurement in cardiomyocyte in the presence of thapsigargin to block SR function. A, Ca2+-influx–dependent Ca2+ transients (after blockage of SERCA2a with 0.5 µmol/L thapsigargin)±10 nmol/L isoproterenol (Iso), F/Fo of Fluo-4 and time scales were shown as inserted bars. B, Normalized data for the effect of Iso±L-NAME on Ca2+ transient amplitude. Compared with male+Iso, *P<0.05.

Figure 7A shows peak I_Ca data versus time in the presence and absence of isoproterenol. As shown in Figure 7B, perfusion of rat cardiomyocytes with 100 nmol/L isoproterenol increased I_Ca amplitude in males to 159±6% of control (n=5) but in females only to 131±8% of control (n=5). Pretreatment with L-NAME had no effect on the isoproterenol-induced I_Ca amplitude in males (177±18%, n=3), but enhanced the increase in I_Ca in the female (177±17%, n=5, P<0.05 versus female+isoproterenol without L-NAME). Again, the male/female difference in I_Ca response to adrenergic stimulation was abolished by L-NAME.

View larger version (26K): [in this window] [in a new window]   Figure 7. ICa measurements in rat cardiomyocytes. A, Representative traces of ICa response to 100 nmol/L isoproterenol in perforated patch with ß-escin. B, Normalized data for the effect of isoproterenol (Iso)±L-NAME on ICa. Average ICa current densities under control conditions (ctr) did not differ between male and female (4.5±0.7 pA/pF vs 5.1±0.3 pA/pF; NS; n=8 and 10) and were unaffected by L-NAME treatment.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Hypercontractile female hearts exhibit less ischemia/reperfusion injury than hypercontractile male hearts.^15,22 As catecholamine levels during ischemia would be greater in an intact animal than a perfused heart, this male/female difference observed with adrenergic stimulation could be very relevant to in vivo conditions during ischemia. Isoproterenol also increases intracellular Ca²⁺, which would activate both eNOS and nNOS, providing an explanation for the observation that the male/female difference is only seen under hypercontractile conditions. Using eNOS^–/– and nNOS^–/– mice, we present the novel finding that both of these NOS isoforms contribute to the protection observed in females. The data in this article also show that females have an increase in eNOS associated with cardiomyocyte caveolin-3. Furthermore, consistent with recent data in the literature showing translocation of nNOS,^30,31 following isoproterenol treatment and ischemia/reperfusion, we observed a net translocation of nNOS to caveolae, which was larger in females than in males. These data provide a mechanism by which both eNOS and nNOS can enhance S-nitrosylation of the L-type Ca²⁺ channel in females. These data and the SNO content data in Table suggest that there may be a threshold level of SNO required to achieve protection. Although association of NOS with caveolin is reported to inhibit basal NOS activity, it has been reported that NOS association with caveolin can localize NOS to the plasma membrane, where on activation, NOS can enhance modification of associated proteins, such as the L-type Ca²⁺ channels.²⁹

To investigate differences in NO generation in males and females of the different genotypes, we examined whether there were male/female differences in SNO under different conditions. Consistent with the observation that only female WT hearts were protected, we only observed a male/female difference in SNO in WT hearts treated with isoproterenol followed by ischemia and reperfusion; no male/female differences were observed in either eNOS^–/– or nNOS^–/– hearts.

To identify potential targets for the NO-induced protection observed in females, we used the biotin switch method²¹ to identify S-nitrosylated proteins in hearts during control perfusion and following ischemia/reperfusion with or without isoproterenol. We made several novel observations. We find that the primary S-nitrosylated protein in a heart membrane fraction is the L-type Ca²⁺ channel/DHPR 1 subunit, which is endogenously S-nitrosylated during normal aerobic perfusion. Furthermore, the S-nitrosylation level of the DHPR 1 is increased after isoproterenol exposure, and when isoproterenol treated hearts are subjected to ischemia and reperfusion, we observe a more robust increase in S-nitrosylation of the DHPR 1 subunit in both males and females of all genotypes. However, consistent with the observation that female protection from ischemia/reperfusion injury in isoproterenol pretreated hearts only occurred in WT hearts (and not in eNOS^–/– or nNOS^–/– hearts), a statistically significant male/female difference in S-nitrosylation of the L-type Ca²⁺ channel was found in only WT hearts.

The majority of data suggest that S-nitrosylation of the L-type Ca²⁺ channel results in a decrease in activity of the channel. For example, addition of isoproterenol to eNOS^–/– hearts has been shown to result in enhanced Ca²⁺ entry through the L-type Ca²⁺ channel compared with WT hearts.⁵ An increase in S-nitrosylation of the L-type Ca²⁺ channel, as occurs in female WT hearts following ischemia/reperfusion in the presence of isoproterenol, would be expected to result in less Ca²⁺ entry into myocytes in females, which would lead to less SR Ca²⁺ loading and less Ca²⁺ induced ischemic injury. Consistent with these data, female myocytes treated with isoproterenol have a lower peak systolic Ca²⁺ and less SR Ca²⁺ (Figure 5), less Ca²⁺ entry (Figure 6), and less of an increase in I_Ca (Figure 7). Furthermore, these male/female differences in systolic Ca²⁺ and SR Ca²⁺ were blocked by L-NAME (Figures 5 through 7).

The data in this article are consistent with the following hypothesis for cardioprotection in females. An increase in Ca²⁺, as occurs with addition of isoproterenol or other hypercontractile conditions, leads to a greater increase in NO production and protein S-nitrosylation in females, because of increased eNOS and nNOS association with caveolin-3 in females under these conditions. The increase in S-nitrosylation of the L-type Ca²⁺ channel in females reduces Ca²⁺ entry and SR loading at the start of ischemia, thereby reducing Ca²⁺ overload during ischemia and reperfusion and thus reducing ischemia/reperfusion injury. Isoproterenol alone increases S-nitrosylation of the L-type Ca²⁺ channel, and it is possible that S-nitrosylation of the L-type Ca²⁺ channel before ischemia may be important to provide protection.

In summary, both constitutive isoforms of NOS play essential roles in the gender differences in ischemia-reperfusion injury under adrenergic stimulation. The correlation between S-nitrosylation of the L-type Ca²⁺ channel/DHPR1, the change in peak systolic and SR Ca²⁺, and the degree of recovery of postischemic contractile function suggest that protein S-nitrosylation plays an important role in cardioprotection. These are the first data to directly demonstrate S-nitrosylation of the L-type Ca²⁺ channel and to show the male/female differences in S-nitrosylation of the L-type Ca²⁺ channel following ischemia/reperfusion after isoproterenol. In addition, the translocation of nNOS from SR to sarcolemma in adrenergically stimulated ischemic/reperfused hearts and the presence of S-nitrosylation of L-type Ca²⁺ channel in eNOS^–/– hearts provide new insights into the regulation of calcium cycling by spatial confinement and translocation of constitutive isoforms of NOS in cardiomyocytes.^1,5,34

	Acknowledgments

 
J.S. and E.M. were supported by the National Institute of Environmental Health Sciences Intramural Program. E.P. was supported by a Postdoctoral Fellowship from the American Heart Association, and D.B. was supported in part by NIH grant HL 30077. C.S. was supported in part by NIH grant HL-39752.

	Footnotes

 
Original received October 26, 2004; resubmission received April 28, 2005; revised resubmission received December 12, 2005; accepted December 21, 2005.

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