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Cardiovascular Research Advance Access originally published online on March 13, 2008
Cardiovascular Research 2008 79(1):179-186; doi:10.1093/cvr/cvn068
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Gender-specific hypertension and responsiveness to nitric oxide in sGC{alpha}1 knockout mice

Emmanuel S. Buys1,2,3,4,{dagger}, Patrick Sips1,2,{dagger}, Pieter Vermeersch5,{dagger}, Michael J. Raher3,4, Elke Rogge1,2, Fumito Ichinose3,4, Mieke Dewerchin5, Kenneth D. Bloch3,4, Stefan Janssens5 and Peter Brouckaert1,2,*

1 Department for Molecular Biomedical Research, Flanders Institute for Biotechnology (VIB), Technologiepark 927, B-9052 Ghent, Belgium
2 Department of Molecular Biology, Ghent University, B-9052 Ghent, Belgium
3 Cardiovascular Research Center, Massachusetts General Hospital, 149, 13th street, Charlestown, MA 02129, USA
4 Anesthesia Center for Critical Care Research, Department of Anesthesia and Critical Care, Massachusetts General Hospital, 50, Blossom Street, Boston, MA 02114, USA
5 Centre for Transgene Technology and Gene Therapy, University of Leuven and VIB, Herestraat 49, B-3000 Leuven, Belgium

* Corresponding author. Tel: +32 9 33 13 710; fax: +32 9 33 13 609. E-mail address: peter.brouckaert{at}dmbr.ugent.be

Received 4 September 2007; revised 5 March 2008; accepted 7 March 2008

Time for primary review: 23 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Aim: The effects of nitric oxide (NO) in the cardiovascular system are attributed in part to cGMP synthesis by the {alpha}1β1 isoform of soluble guanylate cyclase (sGC). Because available sGC inhibitors are neither enzyme- nor isoform-specific, we generated knockout mice for the {alpha}1 subunit (sGC{alpha}1–/– mice) in order to investigate the function of sGC{alpha}1β1 in the regulation of blood pressure and cardiac function.

Methods and results: Blood pressure was evaluated, using both non-invasive and invasive haemodynamic techniques, in intact and gonadectomized male and female sGC{alpha}1–/– and wild-type (WT) mice. Cardiac function was assessed with a conductance catheter inserted in the left ventricle of male and female sGC{alpha}1–/– and WT mice. Male sGC{alpha}1–/– mice developed hypertension (147 ± 2 mmHg), whereas female sGC{alpha}1–/– mice did not (115 ± 2 mmHg). Orchidectomy and treatment with an androgen receptor antagonist prevented hypertension, while ovariectomy did not influence the phenotype. Chronic testosterone treatment increased blood pressure in ovariectomized sGC{alpha}1–/– mice but not in WT mice. The NO synthase inhibitor N{omega}-nitro-L-arginine methyl ester hydrochloride raised blood pressure similarly in male and female WT and sGC{alpha}1–/– mice. The ability of NO donor compounds to reduce blood pressure was slightly attenuated in sGC{alpha}1–/– male and female mice as compared to WT mice. The direct sGC stimulator BAY 41-2272 reduced blood pressure only in WT mice. Increased cardiac contractility and arterial elastance as well as impaired ventricular relaxation were observed in both male and female sGC{alpha}1–/– mice.

Conclusion: These findings demonstrate that sGC{alpha}1β1-derived cGMP signalling has gender-specific and testosterone-dependent cardiovascular effects and reveal that the effects of NO on systemic blood pressure do not require sGC{alpha}1β1.

KEYWORDS Nitric oxide; Gender; Hypertension; Blood pressure; Ventricular function


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Nitric oxide (NO) modulates the function of a variety of physiological systems including host defense, neurotransmission, gastrointestinal motility, genital function, and the cardiovascular system. Endothelial NOS (NOS3)-derived NO plays important roles in maintaining normal vascular tone and blood pressure.1,2 These effects of NO are thought to be mediated by the activation of its principal receptor, soluble guanylate cyclase (sGC), leading to cGMP synthesis and the activation of cGMP-dependent protein kinase I (cGKI).3–5 The catalytically active sGC heterodimers are composed of a common β1 subunit and an {alpha}1 or {alpha}2 subunit. Although the sGC{alpha}1β1 isoform was thought to be critical for smooth muscle relaxation6,7 and was recently shown to be essential for NO-mediated pulmonary vasodilation,8 other studies suggested that low levels of cGMP, generated by sGC{alpha}2β1, are sufficient to mediate the systemic vasodilatory effect of NO.9–11

NO-donor compounds [e.g. nitroglycerin and sodium nitroprusside (SNP)] have long been used to treat a variety of disorders including angina pectoris, congestive heart failure, and hypertension. However, the therapeutic efficacy of these agents is limited by tachyphylaxis and, in the case of SNP, the generation of toxic by-products (cyanide). Recently, a series of drugs have been identified which can directly activate sGC in an NO-independent manner.12 The development of these drugs as novel therapeutic agents has led to a resurgence of interest in the role of sGC in a variety of cardiovascular diseases.

To study the role of sGC{alpha}1 in the regulation of blood pressure and because available sGC inhibitors are neither enzyme- nor isoform-specific,13–16 we generated knockout mice for the {alpha}1 subunit (sGC{alpha}1–/– mice). We report that male, but not female, sGC{alpha}1–/– mice are hypertensive, that the development of hypertension is androgen-dependent and that NO can modulate systemic blood pressure in an sGC{alpha}1β1-independent manner.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
2.1 Generation and molecular characterization of sGC{alpha}1–/– mice
sGC{alpha}1–/– mice with a targeted deletion of the sixth exon were generated using standard methods, as described in detail in the online supplementary data. Methods for immunoblot analysis, quantitative RT–PCR, measurement of sGC enzyme activity, gonadectomy, flutamide treatment, testosterone treatment, and measurement of testosterone levels, are also described in the online supplementary data. Age and strain of the mice used in each experiment is summarized in Table S1 in the online supplementary data. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996).

2.2 Non-invasive blood pressure measurements
Systolic blood pressure (SBP) and heart rate (HR) were measured non-invasively in conscious sGC{alpha}1–/– mice [and their wild-type (WT) littermates] on a mixed Swiss-129 background, using a tail-cuff system warmed at 37°C (Visitech BP-2000 Blood Pressure Analysis System).17 Mice were studied when they were at least 16 weeks of age (except for the age-dependency study where mice were studied at ages 12–18 weeks). Mice were habituated to the blood pressure measurement device for 7–10 days and underwent two cycles of 10 measurements per day for 10 days for blood pressure determination. N{omega}-nitro-L-arginine methyl ester hydrochloride (L-NAME, 100 mg/kg), SNP (1.5 mg/kg), diethylenetriamine NONOate (DETA-NO, 60 mg/kg), or BAY 41-2272 (4 mg/kg), as well as the vehicle controls [phosphate-buffered saline (PBS) or, for BAY 41-2272, 4% DMSO+8% cremophor in PBS], were administered intraperitoneally (ip).

2.3 Invasive haemodynamic measurements
sGC{alpha}1–/– and WT mice on a 129 background between 10 and 13 weeks of age were anaesthetized by ip injection with ketamine (100 mg/kg), fentanyl (50 µg/kg), and pancuronium (2 mg/kg); intubated; and mechanically ventilated (FiO2 = 1, 10 µl/g, 120 breaths per minute). A saline-filled catheter was inserted into the left carotid artery for infusion of saline (2 mL/h) and for measurement of mean arterial blood pressure (MAP). To measure cardiac function, the chest was opened, and a SPR-839 pressure–volume conductance catheter (Millar Instruments, Houston, TX, USA) was introduced through the apex into the left ventricle (LV), as described previously.18,19 HR and LV end-systolic and end-diastolic pressures (LVESP and LVEDP, respectively) were measured. The maximum and minimum first derivative of developed LV pressure (dP/dtmax and dP/dtmin, respectively), the cardiac output (CO), the ejection fraction (EF), the time constant of isovolumic relaxation ({tau}), the total peripheral resistance (TPR= MAP/CO) and the arterial elastance (Ea) were calculated. In a subset of animals, transient occlusion of the inferior vena cava was used to generate the end-systolic pressure–volume relationship from which the end-systolic pressure/volume ratio (Ees) and preload recruitable stroke work (PRSW, the slope of the relationship between stroke work and end-diastolic volume) were determined. After baseline measurements, SNP (5 and 10 µg/kg), spermine NONOate (SPER-NO, 2 µg/kg), or BAY 41-2272 (100 µg/kg) were administered intravenously (iv, 50 µl bolus in the jugular vein), and LVESP measurements were repeated.

Telemetry was performed as described previously.8 Briefly, 10- to 12-week-old male and female, sGC{alpha}1–/– and WT mice on a 129 background, were anaesthetized by ip injection with ketamine (75 mg/kg) and xylazine (10 mg/kg). A transmitter (model TA11PA-C20, Data Sciences International, St Paul, MN, USA) was implanted subcutaneously, and the catheter was introduced in the left carotid artery. Mice were allowed to recuperate for at least 7 days before recordings. SBP, measured continuously for 24 h in male WT and sGC{alpha}1–/– mice, was reported previously.8

2.4 Statistical evaluation
Unless stated otherwise, data were compared using multiple comparisons by two-way ANOVA. After ANOVA, the Bonferroni post hoc test was used to compare separate groups. Data are presented as mean ± SEM. P < 0.05 was considered as significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Supplementary material
 Funding
 References
 
3.1 Generation of sGC{alpha}1-deficient mice
Homozygous mice with a targeted deletion of the sixth exon of the gene encoding sGC{alpha}1 (see Supplementary material online, Figures S1 and S2A) were viable and fertile and displayed a Mendelian distribution in the genotype of offspring of heterozygous breeding pairs (26% WT, 47% sGC{alpha}1+/–, and 27% sGC{alpha}1–/–). sGC{alpha}1–/– mice were indistinguishable from sGC{alpha}1+/– and WT littermates in appearance and behaviour. In sGC{alpha}1–/– mice, a mutant 4.5-kb RNA was detected using RNA blot hybridization and a probe spanning exons 2 to 4 (Supplementary material online, Figure S2B). RT–PCR using primers flanking the sixth exon amplified a WT 650-bp product and a mutant 180-bp product (Supplementary material online, Figure S2C). Immunoblot analysis revealed a mutant 59-kDa sGC{alpha}1-immunoreactive protein in mice homozygous or heterozygous for the mutant allele (Supplementary material online, Figure S2D). These results show that deletion of exon 6, which codes for a conserved portion of the catalytic domain20,21 and whose deletion does not cause a frameshift, resulted in the expression of a mutant protein (sGC{alpha}1{Delta}6) in the sGC{alpha}1–/– mice. When sGC{alpha}1{Delta}6 was co-expressed with sGCβ1 in insect cells, the resulting heterodimer was inactive and could not be stimulated by NO-donor compounds [SNP (Supplementary material online, Figure S3) and 3-morpholinosydnonimine hydrochloride (data not shown)] or direct sGC-stimulators (3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1) and BAY 41-2272) (Supplementary material online, Figure S3). Quantitative RT–PCR experiments indicated the absence of compensatory changes in the levels of mRNAs encoding sGC{alpha}2, sGCβ1, and cGKI{alpha} in aorta (Supplementary material online, Figure S4A and E), as well as in lung, heart, and kidney (Supplementary material online, Figure S4B–E). Similarly, immunoblot techniques did not reveal changes in sGC{alpha}2 or sGCβ1 protein levels in aortae of sGC{alpha}1–/– mice (Supplementary material online, Figure S4F). In lung tissue, however, sGC{alpha}1-deficiency was accompanied by a decrease in the sGCβ1 subunit protein levels (Supplementary material online, Figure S4G). DETA-NO- or BAY 41-2272-induced increases in sGC enzyme activity, observed in aortic, lung, and left ventricular homogenates from both male and female WT mice, were severely attenuated in sGC{alpha}1–/– mice of either gender (Figure 1). In heterozygous mice, DETA-NO-induced increases in sGC enzyme activity were intermediate to those measured in WT and sGC{alpha}1–/– mice, suggesting a gene dose effect and indicating that the mutant sGC{alpha}1 does not function as a dominant negative (Supplementary material online, Figure S5). These results confirm that, although a mutant protein is still expressed in sGC{alpha}1–/– mice, it is catalytically inactive. They also indicate that sGC{alpha}2β1 does not compensate for the loss of cGMP production by sGC{alpha}1β1.


Figure 1
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  		Figure 1 Unstimulated (baseline) and DETA-NO or BAY41-2272- stimulated sGC enzyme activity (expressed as picomoles cGMP produced per mg protein per minute) in aortic (A), lung (B), and left ventricular (C) extracts of 8- to 16-week-old male and female WT (+/+) and sGC{alpha}1–/– (–/–) mice (n for each group is indicated below the respective bar). #P < 0.05 and §P < 0.001 vs. baseline; *P < 0.05 and {dagger}P < 0.001 vs. +/+ samples of the same gender.

 
3.2 Male but not female sGC{alpha}1–/– mice are hypertensive
Using a non-invasive method for measurement of pulse and SBP (‘tail-cuff’), we observed that SBP was higher in male sGC{alpha}1–/– mice (147 ± 2 mmHg) than in WT (125 ± 2 mmHg) or heterozygous mice (129 ± 3 mmHg, Figure 2A, upper panel). Blood pressure did not differ between genotypes in female mice (115 ± 2 mmHg, 119 ± 2 mmHg, and 118 ± 2 mmHg, in sGC{alpha}1–/–, WT, and heterozygous mice, respectively). sGC{alpha}1 deficiency did not affect HR in either male or female mice (Figure 2A, lower panel). These non-invasive tail-cuff measurements, obtained in conscious, restrained mice with a mixed Swiss/129 genetic background, were confirmed using invasive methods in both anaesthetized (Table 1) and conscious, freely moving (Figure 2B, Supplementary material online, Figure S6) 10- to 12-week-old mice on a 129 genetic background. Hypertension detected by tail-cuff in male sGC{alpha}1–/– mice on a mixed Swiss/129 genetic background was age-dependent, developing after 14 weeks of age (Supplementary material online, Figure S7A). However, male sGC{alpha}1–/– mice on a 129 background were hypertensive at 10 weeks of age, suggesting the existence of genetic modifiers of the hypertensive effects of sGC{alpha}1 deficiency. Ageing (10- to 12-month-old) female sGC{alpha}1–/– mice on a 129 background remained normotensive (LVESP: 127 ± 2, n = 4).


Figure 2
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  		Figure 2 Male sGC{alpha}1–/– mice (–/–) develop hypertension. (A) Systolic blood pressure (SBP, upper panel) and heart rate (HR, lower panel) measured using the tail-cuff method in conscious male and female WT mice (+/+, n = 28 for both), sGC{alpha}1+/– mice (+/–, n = 20 and 15, respectively), and –/– mice (n = 22 and 32, respectively) on a mixed Swiss/129 genetic background (at least 16 weeks old). #P < 0.001 vs. all other groups by one-way ANOVA. (B) SBP (upper panel) and HR (lower panel) measured via telemetry in conscious, 10- to 12-week-old unrestrained male and female, +/+ mice (n = 5 and 8, respectively) and –/– mice (n = 8 and 12, respectively) on a 129 background. *P < 0.01 vs. male +/+. 24h SBP values for the male +/+ and –/– mice were reported previously.8

 


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  		Table 1 Left ventricular function in male and female WT and sGC{alpha}1–/– mice

 
To further investigate the mechanisms responsible for the observed gender-specific hypertension, male and female mice of both genotypes were subjected to orchidectomy and ovariectomy, respectively, in order to study the interaction of male and female sex hormones with sGC{alpha}1 deficiency. We did not observe an effect of ovariectomy on blood pressure in either sGC{alpha}1–/– or WT mice on a mixed Swiss/129 genetic background (Figure 3A). However, in contrast to unoperated male sGC{alpha}1–/– mice, orchidectomized male sGC{alpha}1–/– mice, both on a mixed Swiss/129 genetic background (Figure 3B) and on a 129 genetic background (Figure 3C) did not develop hypertension. Total plasma testosterone levels were similar in male sham-operated WT and sGC{alpha}1–/– mice and were greatly reduced in orchidectomized mice of either genotype (Supplementary material online, Table S2). Similarly, the development of hypertension in male sGC{alpha}1–/– mice on a 129 genetic background was prevented by treatment for 5 weeks with the androgen receptor antagonist flutamide (Figure 3C). Neither orchidectomy nor flutamide treatment had an effect on blood pressure in WT mice. However, treating female ovariectomized mice with testosterone, resulting in increased plasma testosterone levels (Supplementary material online, Table S2), markedly increased blood pressure in sGC{alpha}1–/– but not in WT mice (Figure 3D).


Figure 3
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  		Figure 3 Impact of sex hormones on blood pressure in WT and sGC{alpha}1–/– mice. (A) Systolic blood pressure (SBP), serially measured via tail-cuff for 20 weeks after ovariectomy in WT (+/+, solid line, n = 7) and sGC{alpha}1–/– mice (–/–, dashed line, n = 7) on a mixed Swiss/129 background. (B) SBP, measured via tail-cuff in orchidectomized +/+ and –/– mice (n = 6 and 7) and sham-operated male –/– mice (n = 7) on a mixed Swiss/129 genetic background. *P < 0.01 vs. all other groups by one-way ANOVA. (C) SBP, measured using an arterial catheter, of male –/– and +/+ mice on a 129 background, treated with vehicle control (n = 7 and 5, respectively) or flutamide (n = 9 and 4, respectively) and male –/– mice on a 129 background subjected to orchidectomy (n = 5) or sham surgery control (n = 6). *P < 0.01 vs. +/+ vehicle, #P < 0.001 vs. –/– vehicle and §P < 0.001 vs. –/– sham (Student t-test for unpaired observations). (D) SBP, measured using an arterial catheter, of female –/– and +/+ mice on a 129 background, treated with vehicle- (n = 5 and 5, respectively) or testosterone-releasing pellets (n = 5 and 5, respectively). *P < 0.01 vs. all goups.

 
3.3 NO but not BAY 41-2272 modulates blood pressure in sGC{alpha}1–/– mice
The ability of L-NAME, a NOS inhibitor, to increase blood pressure was preserved in both male and female sGC{alpha}1–/– mice and did not differ from that in WT mice (Figure 4A). These non-invasive tail-cuff measurements, obtained in conscious, restrained mice with a mixed Swiss/129 genetic background, were confirmed using telemetry in freely-moving mice on a 129 genetic background (data not shown). Moreover, administration of the NO-donor compounds, SNP and DETA-NO, reduced SBP, measured by tail-cuff, in WT and sGC{alpha}1–/– mice of either gender (Figure 4B, upper panel). Similarly, using invasive methods in anaesthetized mice on a 129 background, SNP and SPER-NO both reduced pressure in WT and sGC{alpha}1–/– mice of either gender (Figure 4B, lower panel). The duration of the response to iv administration of SNP (10 µg/kg), measured invasively, was similarly short-lived in both genotypes, peaking within several seconds (Supplementary material online, Figure S7B). Of note, whereas high doses of SNP (10 µg/kg) reduced LVESP similarly in both male and female WT and sGC{alpha}1–/– mice, a lower dose of SNP (5 µg/kg) decreased LVESP to a lesser extent in sGC{alpha}1–/– than in WT mice of either gender (Figure 4C).


Figure 4
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  		Figure 4 Acute effects on systolic blood pressure (SBP) of compounds acting on the NO/cGMP pathway. (A) L-NAME increased SBP, measured non-invasively in conscious animals 15 min after challenge, to a similar extent in both male and female mice on a mixed Swiss/129 background of either genotype (n = 7 and 5 for male and female WT (+/+), respectively; n = 8 for both male and female sGC{alpha}1–/– (–/–)). *P < 0.01 vs. control, #P < 0.01 vs. +/+. (B) The decrease in blood pressure caused by administration of DETA-NO (60 mg/kg, IP) and SNP (1.5 mg/kg, IP), as measured non-invasively in mice on a mixed Swiss/129 background (upper panel), or SPER-NO (2µg/kg, iv) and SNP (10µg/kg, iv), as measured invasively in mice on a 129 background (lower panel), was similar in all groups (n for each group is indicated above the respective bar). *P < 0.001 vs. PBS control. (C) Effect of SNP administration (5 µg/kg, iv) on LV end systolic pressure (LVESP) in male and female +/+ and –/– mice on a 129 background (n = 5 for all groups). *P < 0.001 and #P < 0.05 vs. +/+ of the same gender.

 
In contrast to observations obtained using NO-donor compounds, the blood pressure-lowering effects of BAY 41-2272, a drug which can stimulate both sGC{alpha}1β1 and sGC{alpha}2β1 in vitro,22 were abolished in sGC{alpha}1–/– mice, as demonstrated using both tail-cuff in mice on a mixed Swiss/129 genetic background (Figure 5, upper panel) and invasive haemodynamic methods in mice on a 129 genetic background (Figure 5, lower panel).


Figure 5
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  		Figure 5 The sGC stimulator BAY 41-2272 decreased systolic blood pressure (SBP, upper panel), as measured non-invasively 15 min after IP injection, in WT (+/+) mice but not in sGC{alpha}1–/– (–/–) mice on a mixed Swiss/129 background (n = 6 for all groups). These findings were confirmed by invasive measurements of LV end systolic pressure (LVESP, lower panel) in mice on a 129 background (n = 5 for all groups). *P < 0.001 vs. vehicle control.

 
3.4 sGC{alpha}1 deficiency is associated with increased vascular resistance, increased cardiac contractility, and impaired ventricular relaxation
LV function was measured invasively in 2- to 4-month-old sGC{alpha}1–/– and WT mice on a 129 background (Table 1). LVESP was higher in male sGC{alpha}1–/– mice than in female sGC{alpha}1–/– mice or in WT mice of either sex. Bodyweight (BW), LV weight-to-BW ratios (LV/BW), HR, LVEDP, and EF, were similar in both genotypes. The maximum rate of developed LV pressure (dP/dtmax), a relatively load-dependent measure of LV systolic function, did not differ significantly between genotypes. However, relatively load-independent measures of LV systolic function, including the Ees and PRSW, were greater in sGC{alpha}1–/– mice than in WT mice of either gender, suggesting that baseline cardiac contractility was enhanced in these mice. Ea and TPR were greater and CO was less in sGC{alpha}1–/– mice than WT mice of either sex. The {tau}, a load-independent measure of LV relaxation, was prolonged in sGC{alpha}1–/– mice suggesting impaired LV relaxation. Together, these results show that increased vascular resistance, increased cardiac contractility, and impaired ventricular relaxation were observed in both male and female sGC{alpha}1–/– mice, albeit to a greater extent in the former.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Supplementary material
 Funding
 References
 
To delineate the role of sGC and, more specifically, the sGC{alpha}1β1 isoform in regulating blood pressure and mediating the haemodynamic effects of NO, we generated mice with a targeted deletion of the catalytic domain of sGC{alpha}1 and characterized their cardiovascular function at baseline and after NO-challenge. We observed gender-specific hypertension in the male sGC{alpha}1–/– mice, both on a mixed Swiss/129 genetic background and on a homogenous 129 genetic background. The hypertension could be prevented by orchidectomy and by treatment with an androgen receptor antagonist. Moreover, chronic testosterone treatment increased blood pressure in ovariectomized sGC{alpha}1–/– mice but not in WT mice. Together, these findings suggest that the interaction of androgens with sGC-signalling modulates blood pressure. The blood pressure-modulating effects of NO-donor compounds and NOS inhibitors but not of the sGC agonist BAY 41-2272, were, at least in part, retained in the absence of functional sGC{alpha}1β1.

Increased Ea and TPR were observed in both male and female sGC{alpha}1–/–, suggesting that sGC{alpha}1 deficiency is associated with increased vascular resistance. The marked reduction in CO, possibly in response to increased vascular resistance, was accompanied by impaired LV relaxation, as reflected by prolonged {tau}. Of note, LV systolic function, assessed using relatively load-independent ejection phase parameters, was increased in both male and female sGC{alpha}1–/– mice as compared to WT mice, suggesting that sGC{alpha}1 modulates myocardial function in mice. This observation is consistent with the previously reported negative inotropic effects of cGMP.23 Additional studies are necessary to further characterize the cardiac phenotype associated with sGC{alpha}1–/– deficiency and to study the impact of gonadectomy on cardiac function in sGC{alpha}1–/– mice.

The mechanisms responsible for the gender-specific effects of sGC{alpha}1 deficiency on blood pressure are uncertain. Importantly, although male but not female sGC{alpha}1–/– mice develop hypertension, a low dose of SNP decreased LVESP to a lesser extent in sGC{alpha}1–/– than in WT mice of either gender. Also, the NO-induced relaxation of aortic rings,11 increase in aortic cGMP levels11 and increases in sGC enzyme activity were similarly attenuated in both male and female sGC{alpha}1–/– mice. Together, these findings suggest that, although impairments in the haemodynamic response to NO were noted in sGC{alpha}1–/– mice, they do not seem to be responsible for the observed gender dimorphism. Prior studies in one strain of NOS3-deficient mice revealed that males have hypertension and females do not,1,24 suggesting that NO/cGMP signalling has a greater role in regulating blood pressure in male than in female mice. However, in an independently generated NOS3–/– strain,2 both male and female mice were hypertensive. Recent studies have indicated that the endothelium-derived hyperpolarization factor system plays a greater role in maintaining normal blood pressure in female than in male mice.24 These findings suggest that male and female mice may have differing capacities to compensate for the hypertensive effects of sGC{alpha}1 deficiency and/or that female mice rely less on sGC{alpha}1 to regulate blood pressure.

The importance and clinical relevance of gender differences in cardiovascular pathology and the underlying molecular pathogenic mechanisms are increasingly recognized and prompt scientists and clinicians to consider gender-specific treatment for cardiovascular diseases.25 Gender-specific cardiovascular differences are often attributed to the protective effects of estrogen and gradually decrease after menopause. More specifically, increased production of NO is thought to play a prominent role in the vasodilator effects of estrogen.26,27 However, ovariectomy did not result in increased blood pressure in either sGC{alpha}1–/– or WT female mice. Orchidectomy, on the other hand, had no effect on blood pressure in WT mice but prevented the development of hypertension in male sGC{alpha}1–/– mice suggesting that the hypertensive effects of sGC{alpha}1 deficiency are mediated by testosterone. The role of androgens in the hypertension associated with sGC{alpha}1 deficiency was confirmed by the observation that treating male sGC{alpha}1–/– mice with the androgen receptor antagonist, flutamide, inhibited the development of hypertension. Ovariectomy, orchidectomy, and flutamide treatment were shown previously not to have an effect on blood pressure in WT mice.28,29 Similar observations were described in spontaneously hypertensive rats: castration at a young age or chronic treatment with flutamide attenuated the development of hypertension in spontaneously hypertensive rats, indicating that hypertension in these rats was dependent on the androgen receptor.30 Moreover, we found that testosterone treatment induced hypertension in female ovariectomized sGC{alpha}1–/– mice but not in WT mice suggesting that the co-existence of sGC{alpha}1 deficiency and androgens is necessary and sufficient to cause hypertension in mice. The mechanisms responsible for the impact of androgens on blood pressure in these animal models remain to be elucidated.

Our observations clearly show that NO can modulate haemodynamic parameters independent of sGC{alpha}1. NOS inhibition and NO-donor compounds increased and decreased blood pressure, respectively, in sGC{alpha}1–/– mice. These findings differ from those in cGKI–/– mice where blood pressure drops in response to SNP were almost abolished.31,32 Interestingly, hypertension was reported in 3- to 4-week-old cGKI–/– mice blood pressure but not in adult cGKI–/– mice.31,32

During preparation of this manuscript, results were published characterizing sGC{alpha}1–/–, sGC{alpha}2–/–, and sGCβ1–/– mice.9,10 Mergia et al.10 reported that chronic administration of L-NAME raised blood pressure similarly in sGC{alpha}1–/– and WT mice, which is in agreement with our findings. S-nitroso glutathione, a NO-donor compound, dilated aortic rings from sGC{alpha}1–/– mice to a lesser extent than those from WT mice, and NO-mediated dilation of sGC{alpha}1–/– aortic rings was blocked by the sGC inhibitor, ODQ. Friebe et al.9 showed that NO-induced relaxation was completely abolished in aortae from sGCβ1–/– mice, and that glyceryl trinitrate failed to decrease SBP in sGCβ1–/– mice. Our data furthermore show that the effects of BAY 41-2272 and low doses of SNP on blood pressure were abolished and attenuated, respectively, in the sGC{alpha}1–/– mice. Together, these studies suggest that sGC{alpha}1 accounts for only part of the hypotensive effects of NO and that a very small increase in sGC activity, by default derived from sGC{alpha}2, is sufficient to mediate NO-induced decreases in blood pressure in sGC{alpha}1–/– mice. In addition, these data show that sGC{alpha}2 is not able to mediate BAY 41-2272-induced decreases in blood pressure in sGC{alpha}1–/– mice, implying that BAY 41-2272 is an isoform-specific sGC{alpha}1β1 stimulator in vivo in mice. Importantly, although sGC{alpha}1 is not essential for NO-mediated systemic vasodilation, it is required for NO-mediated pulmonary vasodilation,8 suggesting that sGC{alpha}2 can not compensate for sGC{alpha}1 deficiency in the pulmonary vasculature and highlighting the existence of vascular bed-specific pathways by which NO mediates vasodilation.

There are several important differences between our observations and those reported by Mergia et al.10 and Friebe et al.9 We observed that male sGC{alpha}1–/– mice are hypertensive, whereas female sGC{alpha}1–/– mice are not. In the reports by Mergia et al.10 and Friebe et al.,9 no separate male and female data were shown. sGC{alpha}1–/– mice were only modestly hypertensive in the study of Mergia et al.10 (111 ± 2 and 104 ± 2 mmHg in sGC{alpha}1–/– and WT mice, respectively), while we report a more marked hypertensive effect of sGC{alpha}1 deficiency (147 ± 2 and 125 ± 2 mmHg in sGC{alpha}1–/– and WT mice, respectively). In the study by Mergia et al.,10 the phenotype of sGC{alpha}1–/– mice on a mixed 129/C57BL6 genetic background was investigated. In our study, blood pressure data were acquired from mice on a mixed Swiss/129 genetic background and on a homogenous 129 genetic background. The existence of genetic modifiers likely explains the differences in the cardiovascular phenotype between sGC{alpha}1–/– mice on different genetic backgrounds.

In conclusion, our observation that male but not female sGC{alpha}1–/– mice are hypertensive, strongly suggests that sGC{alpha}1β1-derived cGMP signalling has gender-specific and testosterone-dependent cardiovascular effects. Furthermore, we demonstrate that NO can modulate blood pressure independently of sGC{alpha}1β1. Our results suggest that NO-independent sGC activators/stimulators, under development for the treatment of hypertension and other cardiovascular diseases, may have differing efficacy in males and females. Clinical trials evaluating these agents should consider the possibility of gender-specific responses.


   Supplementary material
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
Funding
 References
 
Supplementary material is available at Cardiovascular Research online.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
References
 
Research was supported by the Fonds voor Wetenschappelijk Onderzoek (FWO)-Vlaanderen, the Interuniversity Attraction Poles (IUAP) and the Bijzonder Onderzoeksfonds (BOF)-Geassocieerde Onderzoeks Actie (GOA). E.B. was supported by an award from the Northeast Affiliate Research Committee of the American Heart Association, F.I. by the United States Public Health service grant GM079360, and K.D.B. by the United States Public Health service grant HL070896. P.S. is an Institute for the promotion of Innovation by Science and Technology in Flanders (IWT) fellow. P.V. is a research assistant and S.J. a clinical investigator of the FWO-Vlaanderen. S.J. holds a chair supported by Astra-Zeneca.


   Acknowledgements
 
The authors would like to thank the DMBR animal caretakers for maintaining the animal facility, Amanda Graveline, Robrecht Thoonen, Robert Searles, and Kristen Rauwerdink for assistance with experiments.

Conflict of interest: none declared.


   Notes
 
{dagger} These authors contributed equally to this work Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 

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