| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Medicine and Clinical Science (Y.L., I.K., Y.S., M.H., K.K., T.I., I.H., N.T., R.K., K.T., Y.N., M.N., Y.A., K.N.), Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan; Howard Hughes Medical Institute and Department of Pharmacology (D.L.G.), University of Texas, Southwestern Medical Center at Dallas, Dallas, Texas 75390; and Center for Tsukuba Advanced Research Alliance (A.F.), Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8571, Japan
Address all correspondence and requests for reprints to: Yoshihiko Saito, First Department of Internal Medicine, Nara Medical University, 840, Shijo-cho, Kashihara, Nara 634-8522, Japan. E-mail: yssaito{at}naramed-u.ac.jp.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Mice lacking guanylyl cyclase A (GC-A), a natriuretic peptide receptor, exhibit salt-resistant hypertension, myocardial hypertrophy and interstitial fibrosis, and sudden death (before the age of 6 months) (19, 20). In the present study, we found that male GC-A knockout (KO) mice show more pronounced cardiac hypertrophy and fibrosis compared with female GC-A KO mice and that gender-related differences are not seen in wild-type (WT) mice. Additionally, we found that these gender-related differences are attenuated either by castration or flutamide, an androgen receptor (AR) antagonist, and abolished by genetic disruption of angiotensin (Ang) II type 1A (AT1A) receptors in male GC-A KO mice. We propose that androgens contribute to gender-related differences in cardiac structure and that the AT1A receptor and GC-A are involved in a reciprocal fashion.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Measurement of heart rate (HR) and SBP
HR and SBP were measured in conscious mice using a computerized tail-cuff method (Softron Co., Ltd., Tokyo, Japan) (19, 21). Briefly, mice were restrained in a pocket and warmed at 38 C. HR and SBP were measured at 1000–1400 h and calculated as the average of six sessions per day after mice were adapted to the apparatus for 5 d. The validity of this system has been established previously in our laboratory (22).
Measurement of left ventricular weight (LVW) and interstitial fibrosis
After animals were killed by cervical dislocation under anesthesia with ether at 16 wk of age, the hearts were dissected out, LVW was measured, and its ratio to BW (LVW/BW) was calculated and used as an index of ventricular hypertrophy. The left ventricles were then fixed in 10% formalin and prepared for routine histological examination. To determine the degree of collagen fiber accumulation, we randomly selected 20 fields in three individual sections and calculated the ratio of the areas of van Gieson-stained interstitial fibrosis to the total left ventricular area using image analysis software and a Zeiss KS400 system; perivascular fibrosis was excluded in the present study.
mRNA analysis
Total mRNA was prepared from the left ventricle using TRIzol (Life Technologies Inc., Rockville, MD). Expression of mRNAs encoding atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), collagens I and III, TGF-ß1, TGF-ß3, angiotensinogen (Agt), and Ang converting enzyme (ACE) was evaluated using quantitative RT-PCR in a 7700 sequence detector (ABI PRISM, Applied Biosystems, Foster City, CA). The oligonucleotide primers are shown in Table 1
. Glyceraldehyde-3-phosphate dehydrogenase mRNA was also amplified with specific primers and probe (Applied Biosystems).
|
To evaluate the involvement of estrogen in gender-related differences, we compared the phenotypes of sham-operated and ovariectomized (OVX) mice (n = 7–9 per group). Under anesthesia with ether, the ovaries of 10-wk-old female mice were exteriorized, ligated, and removed via bilateral paralumbar incisions, which were then closed with sutures. In sham mice, the ovaries were exteriorized and replaced, and the incisions were closed. Six weeks later, HR and SBP were measured, and the animals were killed.
To investigate the effects of androgens, male mice at 10 wk of age (n = 7–9 per group) were castrated using the trans-scrotal approach. Sham castration consisted of exteriorizing and replacing the testes. As in females, 6 wk later, HR and SBP were measured, and the animals were killed.
To confirm the role of androgen, we ovariectomized female WT and GC-A KO mice (n = 6–7 per group) under anesthesia with ether at 10 wk of age and sc implanted a testosterone pellet (25.0 mg/pellet, 60-darelease, catalog item SA-151) or vehicle pellet (placebo for testosterone, catalog item SC-111) (Innovative Research of America, Sarasota, FL) between the shoulders. Six weeks later, the animals were killed after HR and SBP were measured.
We further confirmed the role of androgens by chronically blocking AR with flutamide (23, 24). Flutamide (Sigma Chemical Co., St. Louis, MO; 8 mg/kg·d, dissolved in polyethylene glycol 300) was sc infused for 6 wk using an osmotic mini-pump (model 2002, Alza Corp., Mountain View, CA) at 10 wk of age in male animals (n = 7–9 per group). The mini-pumps were sc implanted under the mice were anesthetized with ether and changed with new ones every 2 wk. Control mice were administered only vehicle. Six weeks later, the animals were analyzed.
To assess the involvement of the AT1A receptors in GC-A disruption-induced gender difference, we deleted AT1A receptor by the described method above. At 16 wk of age, the animals (n = 5–9 per group) were analyzed.
To further support the conclusion of AT1A receptor involvement, we castrated male double-KO mice (10 wk old; n = 5–6 per group) and chronically infused exogenous testosterone and analyzed the animals by the described methods above.
Statistical analysis
All results are expressed as means ± SEM. Data were analyzed by one-facter ANOVA. If a statistically significant effect was found, the Newman-Keuls test was performed to isolate the difference between the groups. Values of P < 0.05 were considered statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). In contrast, in WT mice, there was no difference in LVW/BW ratio in males vs. females (Fig. 1A). In addition, male GC-A KO mice, but not WT mice, exhibited higher levels of left ventricular fibrosis than did females (378% vs. 44%, respectively; Fig. 1, B and C). On the other hand, there was no gender-related difference in HR (WT female 599.6 ± 26.1 vs. male 619.0 ± 52.4 beats/min; GC-A KO female 574.5 ± 25.9 vs. male 571.3 ± 28.1 beats/min; n = 7–9 per group) or in SBP (WT female 118.4 ± 1.7 vs. male 113.2 ± 3.4 mm Hg; GC-A KO female 140.0 ± 3.4 vs. male 147.4 ± 2.2 mm Hg; n = 7–9 per group) in either genotype. Male mice weighed more than females, but there was no difference between genotypes [WT female 25.0 ± 0.9 vs. male 32.3 ± 1.6 g (P < 0.05); GC-A KO female 25.1 ± 0.8 vs. male 31.6 ± 1.9 g (P < 0.05); n = 7–9 per group].
|
, A–C), or BW (WT sham 25.0 ± 0.9 vs. OVX 25.6 ± 0.8 g; GC-A KO sham 25.1 ± 0.8 vs. OVX 25.1 ± 1.6 g; n = 7–9 per group).
Neither castration nor administration of an AR antagonist diminishes cardiac hypertrophy and fibrosis in male GC-A KO mice, whereas testosterone infusion increases cardiac hypertrophy and fibrosis in OVX GC-A mice
In contrast to OVX, removal of testes was associated with a marked reduction in the LVW/BW ratio and in ventricular fibrosis in GC-A KO mice (by 20.5 and 44.7%, respectively) but not to levels comparable to those seen in WT mice. Castration had no effect in WT mice (Fig. 1). Castration reduced BW in male WT as well as in male GC-A KO mice]WT 5.7 ± 0.5 vs. 2.1 ± 0.4 g (P < 0.05), and GC-A KO 6.6 ± 0.9 vs. 2.7 ± 0.6 g (P < 0.05) for sham and castrated groups, respectively; n = 7–9 per group]. Castration had no effect on HR (WT 619.0 ± 52.4 vs. 606.2 ± 45.9 beats/min, and GC-A KO 571.3 ± 28.1 vs. 600.2 ± 13.2 beats/min, for sham and castrated groups, respectively; n = 7–9 per group) or SBP (WT 113.2 ± 3.4 vs. 105.8 ± 4.2 mm Hg, and GC-A KO 147.4 ± 2.2 vs. 142.2 ± 6.3 mm Hg, for sham and castrated groups, respectively; n = 7–9 per group). The AR antagonist flutamide had similar effects to castration on LVW/BW, fibrosis (Fig. 2), HR (data not shown), and SBP (data not shown). In contrast, chronic infusion of testosterone increased LVW/BW ratio (by 20%) and cardiac fibrosis (by 114%) in OVX GC-A mice but not in OVX WT mice (Fig. 3). Testosterone treatment was also associated with increased BW in GC-A KO but not in WT mice ]WT 2.6 ± 0.3 vs. 3.2 ± 0.3 g (P < 0.05), and GC-A KO 2.9 ± 0.2 vs. 5.2 ± 0.5 g (P < 0.05), for sham and testosterone-treated groups, respectively; n = 6–9 in each group[. HR and SBP were not affected by testosterone treatment (data not shown).
|
|
). Again, no gender-related difference or castration-associated effects were seen in WT mice (Fig. 4). In contrast to the above mentioned genes, the levels of Agt and ACE mRNAs were higher in males than in females, and castration of males strongly suppressed their expression, and their levels were comparable in both genotypes of mice.
|
).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Next, we examined the effects of androgens. ARs are widely distributed in the cardiovascular system, where they have been identified on aortic, peripheral vascular, ventricular, and atrial myocytes (30), and were recently shown to mediate robust, testosterone-induced hypertrophic responses in cardiac myocytes (12). Nevertheless, although virtually all men have much higher levels of androgens than women do, not all men exhibit more severe cardiac hypertrophy and fibrosis. In the present study, significant gender-related differences in cardiac abnormalities were observed only in GC-A KO mice. In addition, it is notable that both castration and AR antagonist markedly diminished cardiac hypertrophy and fibrosis in male GC-A KO mice, and chronic testosterone infusion increased cardiac mass and fibrosis only in OVX GC-A KO mice. ANP and BNP are well established molecular markers for cardiac hypertrophy. It has been demonstrated that the testosterone metabolite dihydrotestosterone is able to increase ANP secretion from ventricular myocytes. An AR antagonist, cyproterone, abolished this effect (12). Like cardiac geometric changes, changes in ANP and BNP, or markers for ventricular fibrosis, collagen I, and collagen III were higher in male GC-A KO mice than in females and were reduced by removal of testes. These findings suggest that androgens play an important role in gender-related cardiac differences in GC-A KO mice. Castration in males and AR antagonist could not reduce the cardiac mass and fibrosis to WT levels, suggesting that more than androgens are involved.
Ang II is known to potently stimulate cardiomyocyte and fibroblast growth, both in vitro and in vivo (31, 32), and the tissue renin-Ang system is known to play a key role in cardiac remodeling (33). It has been proposed that increased ACE abundance in the hypertrophied and failing heart may contribute to the local generation of Ang II and impact cardiac remodeling through local paracrine or autocrine effects (34, 35, 36). The greater abundance of ventricular ACE in males may contribute to the tendency of male rodents to develop cardiac abnormalities, which has been described in transgenic mouse models (37, 38) and spontaneously hypertensive rats (39) and in response to left ventricular pressure overload in rats (10) and in humans (40, 41, 42). It has been reported that hepatic Agt mRNA levels are higher in intact male hypertensive rats than in the females; moreover, those levels are reduced by orchidectomy and increased by administration of testosterone (43). Recently, Freshour et al. (44) demonstrated a gender difference in the expression of ACE in the murine heart with greater cardiac ACE levels seen in male animals compared with females. Moreover, ventricular ACE levels were substantially decreased in androgen-deprived males (44). Consistent with those reports, our data show that levels of cardiac Agt and ACE expression are higher in the ventricles of both GC-A KO and WT males than they are in females. Castration reduced expression of Agt in the male ventricle to levels approximating those seen in the females in both WT and GC-A KO mice. Given the evidence that Ang II has hypertrophic and fibrogenic activities in the heart, Ang II is a possible candidate to link androgens with cardiac abnormalities. It should be noted, however, that gender-related increases in LVW/BW ratios and interstitial fibrosis were observed only in the GC-A KO mice but not in WT mice, despite the similar up-regulation of Agt and ACE expression in ventricles of both genotypes of mice. We recently observed that GC-A signaling counteracts Ang II-induced cardiac abnormalities. A subpressor dose of Ang II increased cardiac mass and fibrosis only in male GC-A KO but not WT mice, suggesting an augmented responsiveness to Ang II in the heart of GC-A KO mice (22). Thus, we speculate that gender-related differences in the heart were made manifest by lacking inhibitory actions of GC-A on AT1 signaling in GC-A KO mice. Inhibitory effects of GC-A were also supported by the overexpression of TGF-ß1 and -ß3, which are activated by AT1A signaling and responsible for interstitial fibrosis (28, 45, 46, 47), in GC-A KO mice.
To further test this hypothesis, we deleted the AT1A receptor gene, which mediates classical Ang II actions, including cardiac hypertrophy and fibrosis, by crossing GC-A KO mice and AT1A KO mice. The gender-related cardiac differences were absent in the double-KO mice. Furthermore, castration of males did not reduce and testosterone administration failed to increase the cardiac mass and fibrosis in male double-KO mice. These results strongly suggest that GC-A prevents androgen-induced cardiac abnormalities mainly by inhibiting the androgen-Ang II-AT1A axis.
The present data did not indicate that androgens and Ang II solely provide a causative contribution to gender-related cardiac differences in GC-A KO mice. LVW/BW in male GC-A KO mice after castration or flutamide treatment were comparable to that in female GC-A KO mice, but the degree of fibrosis was still higher in male GC-A KO mice after the treatments than in females, suggesting androgens mostly contribute to gender-related left ventricular hypertrophy, and at least approximately 50% to the gender-related increase in fibrosis. In the case of AT1A blockade, LVW/BW in GC-A KO mice were reduced to the level corresponding to that in WT mice, in which there was no difference in hypertrophy, and fibrosis was more intensively reduced by knocking out the AT1A receptor, compared with blockade of ARs. These findings suggest that AT1A signaling contributes not only to gender-related cardiac abnormalities but also to abnormalities specifically observed in both genders of GC-A KO mice, suggesting androgen is one of the factors up-regulating the Ang system. Additional studies are required to elucidate the entire mechanism for gender-related difference observed in GC-A KO mice, in which other molecules, such as catecholamines and endothelin, would be involved.
Another interesting finding is that castration or the treatment with androgen antagonist improved cardiac abnormalities in GC-A KO mice without significant change in SBP. As mentioned above, a subpressor dose of Ang II exaggerated hypertrophy and fibrosis in GC-A KO mice (22). It is likely, therefore, that androgen-induced Ang II is sufficient for inducing hypertrophy and fibrosis in GC-A KO mice but not to elevate BP.
The presence or absence of androgens in male GC-A KO mice showed marked effects on the expression levels of ANP. The molecular mechanism is unclear at present. In the present study, despite the similar up-regulation of Agt and ACE expression in ventricles of both male WT and GC-A KO mice, ANP was markedly increased only in male GC-A KO mice. We demonstrated a similar expression level of AT1A mRNA in males and females of both WT and GC-A KO mice (data not shown). Therefore, it seems that androgen-induced intracellular signaling at a postreceptor level for modulation of ANP gene expression is up-regulated in GC-A KO mice. Ang II is known to increase ANP expression mediated by protein kinase C or MAPK (48). Further examination is necessary to determine whether the protein kinase C or MAPK pathway is involved in the elevation of ANP by androgens.
Recently, Nakayama et al. (49) described a functional mutation in the 5'-flanking region of the human GC-A gene that reduces transcriptional activity by more than 70% in reporter gene assay, is present in approximately 5% of the hypertensive individuals in Japan, and is associated with cardiac hypertrophy. Evidence also suggests that GC-A receptors may be down-regulated in patients with chronic, severe heart failure (50). Indeed, there may be substantial numbers of patients whose abnormal GC-A signaling makes them susceptible to androgen-induced cardiac abnormalities. From the clinical points of view, the present study raises the possibility of the prophylactic use of Ang II receptor blocker or AR antagonist in patients with loss of functional mutations in the GC-A gene.
Taken together, our findings strongly support the hypothesis that androgen contributes to cardiac abnormalities via the AT1A receptor. Furthermore, this androgenic effect is normally inhibited by stimulation of GC-A by natriuretic peptides.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Abbreviations: ACE, Angiotensin converting enzyme; Agt, angiotensinogen; Ang, angiotensin; ANP, atrial natriuretic peptide; AR, androgen receptor; AT1A, Ang II type 1A; BNP, brain natriuretic peptide; BW, body weight; GC-A, guanylyl cyclase-A; HR, heart rate; KO, knockout; LVW, left ventricular weight; OVX, ovariectomy; SBP, systolic blood pressure; WT, wild-type.
Received June 30, 2003.
Accepted for publication October 22, 2003.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-reductase activity and androgen-dependent proliferation of vascular smooth muscle cells. J Steroid Biochem Mol Biol 50:169–174[CrossRef][Medline]
1-adrenergic blockade, converting enzyme inhibitor therapy, and angiotensin II subtype 1 receptor blockade on ventricular remodeling in the dog. Circulation 90:3034–3046
This article has been cited by other articles:
 |
|
 |
|
  E. S. Buys, P. Sips, P. Vermeersch, M. J. Raher, E. Rogge, F. Ichinose, M. Dewerchin, K. D. Bloch, S. Janssens, and P. Brouckaert Gender-specific hypertension and responsiveness to nitric oxide in sGC{alpha}1 knockout mice Cardiovasc Res, July 1, 2008; 79(1): 179 - 186. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. E. Haddad, L. J. Saunders, S. D. Crosby, M. Carles, F. del Monte, K. King, M. R. Bristow, F. G. Spinale, T. E. Macgillivray, M. J. Semigran, et al. Human cardiac-specific cDNA array for idiopathic dilated cardiomyopathy: sex-related differences Physiol Genomics, April 1, 2008; 33(2): 267 - 277. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. R. Forte Jr. Atriopeptins: Protection from Myocardial Hypertrophy and Heart Failure Endocrinology, September 1, 2007; 148(9): 4160 - 4161. [Full Text] [PDF]
|
|
|
|
 |
|
 C. F. Deschepper and B. Llamas Hypertensive Cardiac Remodeling in Males and Females: From the Bench to the Bedside Hypertension, March 1, 2007; 49(3): 401 - 407. [Full Text] [PDF]
|
|
|
|
 |
|
 L. J Ellmers, N. J A Scott, J. Piuhola, N. Maeda, O. Smithies, C. M Frampton, A M. Richards, and V. A Cameron Npr1-regulated gene pathways contributing to cardiac hypertrophy and fibrosis J. Mol. Endocrinol., February 1, 2007; 38(2): 245 - 257. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. L. Rogers, A. R. Mitchell, C. Maric, K. Sandberg, A. Myers, and S. E. Mulroney Effect of sex hormones on renal estrogen and angiotensin type 1 receptors in female and male rats Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2007; 292(2): R794 - R799. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. D. Lekgabe, S. G. Royce, T. D. Hewitson, M. L. K. Tang, C. Zhao, X. L. Moore, G. W. Tregear, R. A. D. Bathgate, X.-J. Du, and C. S. Samuel The Effects of Relaxin and Estrogen Deficiency on Collagen Deposition and Hypertrophy of Nonreproductive Organs Endocrinology, December 1, 2006; 147(12): 5575 - 5583. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Souzeau, B. Llamas, S. Belanger, S. Picard, and C. F. Deschepper A Genetic Locus Accentuates the Effect of Volume Overload on Adverse Left Ventricular Remodeling in Male and Female Rats Hypertension, January 1, 2006; 47(1): 128 - 133. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Ikeda, K.-i. Aihara, T. Sato, M. Akaike, M. Yoshizumi, Y. Suzaki, Y. Izawa, M. Fujimura, S. Hashizume, M. Kato, et al. Androgen Receptor Gene Knockout Male Mice Exhibit Impaired Cardiac Growth and Exacerbation of Angiotensin II-induced Cardiac Fibrosis J. Biol. Chem., August 19, 2005; 280(33): 29661 - 29666. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Wakefield, V. Cornish, F. Broackes-Carter, and E. Sim Arylamine N-acetyltransferase 2 Expression in the Developing Heart J. Histochem. Cytochem., May 1, 2005; 53(5): 583 - 592. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X.-J. Du Gender modulates cardiac phenotype development in genetically modified mice Cardiovasc Res, August 15, 2004; 63(3): 510 - 519. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
