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Baker Heart Research Institute and Alfred Heart Centre, Alfred Hospital, Melbourne, Victoria 8008, Australia
Address all correspondence and requests for reprints to: Xiao-Jun Du, Baker Medical Research Institute, PO Box 6492, St Kilda Road Central, Melbourne, Victoria 8008, Australia. E-mail: xiaojun.du{at}baker.edu.au.
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Abstract |
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Introduction |
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There is good evidence for direct cardiac protection by estrogen in different species (6, 14, 15). Estrogen protects hearts against necrotic and apoptotic cell death and fibrosis (16, 17, 18) and attenuates myocardial hypertrophy and left ventricular (LV) remodeling (3, 6, 13, 14). However, recent clinical trials have failed to demonstrate reduction in cardiovascular events in postmenopausal women receiving estrogen replacement therapy (19, 20). In contrast to estrogen, the role of androgen has been little studied, but there is evidence that androgen up-regulates expression of hypertrophy-related genes (6, 21, 22).
Activation of the ß-adrenergic system in the setting of cardiac disorders has been implicated as contributing to the development of heart failure. Transgenic (TG) strains with overexpression of the ß1-adrenergic receptor (AR) (23, 24), the ß2-AR (at high levels) (25, 26), Gs
, or protein kinase A (27), but not strains overexpressing ß-AR kinase inhibitor or adenylyl cyclases (28, 29, 30, 31), have been shown to develop cardiomyopathy and heart failure with aging. Following the previous demonstration of a cardiomyopathic phenotype in a ß2-AR TG line (26), we hypothesized that sex differences exist in this cardiomyopathic strain and that estrogen, and possibly androgen, are responsible for such sex differences. We have therefore studied male and female TG mice with cardiac overexpression of ß2-AR and their wild-type (WT) littermates to explore these two hypotheses.
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Materials and Methods |
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Hemodynamic measurement and ß2-AR binding assay
Cardiac catheterization was performed in a separate group of 6-mo-old male (n = 6) and female (n = 7) TG mice. Animals were anesthetized (mixture of ketamine/xylazine/atropine at 60/12/0.9 mg/kg, respectively) and a 1.4 F Millar catheter inserted into the LV via the right carotid artery. Heart rate, LV systolic pressure, and the maximal rates of rise and fall in LV pressure (dP/dtmax and dP/dtmin) were recorded digitally. The LV was dissected, frozen, and used for ß2-AR binding assay as described previously (26).
Echocardiography
Echocardiography was performed using an Agilent Sonos 5500 machine and a 15-MHz linear transducer, as described previously (33). Mice were lightly anesthetized (the same mixture as for the hemodynamic study). Two-dimensional (2-D) image-guided M-mode trace crossing the LV was recorded digitally. The following parameters were derived from the M-mode tracings (33): LV end-systolic and end-diastolic diameters (LVESd, LVEDd), external LV diastolic diameter (ExLVDd), ventricular wall thickness at systole (WTs), and diastole (WTd), fractional shortening [FS% = (LVEDd - LVESd)/ LVEDd], wall-thickening index [WTI = (WTs - WTd) /WTd], LV mass [= (ExLVDd3 - LVEDd3) x1.055], and the ratio of LVEDd and WTd.
Morphological examination
Autopsy was performed in all animals either after animals were found dead or were killed when signs indicating critical illness were observed or at the end of the 15-mo study period. The chest was opened to inspect for the presence of pleural effusion and hearts were then excised and weighed after removal of blood clots. The presence of organized thrombus in the left atrium was determined by its yellowish color and tight adhesion to the atrial wall. The weight of thrombus was subtracted from heart weight. Lung weight was measured and pulmonary congestion was considered to be present if a lung weight exceeded the value of mean + 2 SD from male (241 mg) and female (233 mg) WT mice.
Myocyte size, collagen content, and terminal transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining
Hearts from mice either found dead or killed at the end of the study were fixed in 10% formalin in PBS, embedded in paraffin, and serially cut from the apex to the base. Transverse sections (5 µm, six sections per heart) were collected and stained with hematoxylin and eosin or with 0.1% picrosirius red. Microscopic images were gathered with a charge-coupled device video camera and analyzed with Optimas 6.2 image analysis software (Media Cybernetics, Silver Spring, MD). Myocyte cross-sectional areas were measured from 10 randomly selected fields (total 70–100 cells per LV). Ventricular interstitial collagen content was quantified from picrosirius red-stained serial sections. Data were expressed as averaged percentage of 60 fields.
To determine the frequency of apoptotic cell death, separate groups of mice were killed at 5 or 15 mo of age, and the number of myocyte nuclei in the LV were manually counted in the section obtained from the equator of the LV. DNA fragmentation of LV myocytes was evaluated by the TUNEL method. A commercial kit (Roche Molecular Biochemicals, Mannheim, Germany) was used according to the manufacturer’s instruction. Images were obtained digitally with a video camera and the numbers of negative and brown-stained TUNEL-positive nuclei of myocytes were manually counted (about 4000–8000 cells per section) across the entire section at x40 objective magnification, in a blinded manner.
Hormone determination
To validate the reduction in gonadal hormones by gonadectomy, plasma samples were collected from 15-mo-old TG and WT intact or gonadectomized mice. We used commercially available competitive immunoassays to measure plasma levels of progesterone (Vitros, Ortho-Clinical Diagnostics, Amersham, Buckinghamshire, UK) and testosterone (Diagnostic Products Co., Los Angeles, CA). The detecting limit is 2 nmol/liter for progesterone and 1.7 nmol/liter for testosterone.
Expression of -myosin heavy chain (MHC) and TGF-ß1
Total RNA was isolated from the LV by the acid guanidinium-phenol-chloroform method and quantified by absorbance measurement at 260/280 nm. -MHC mRNA levels were determined by solution hybridization/ribonuclease protection assay as previously described (7, 34). Transforming growth factor ß1 (TGF-ß1) mRNA levels were determined by Northern blotting as previously reported (35). Levels of -MHC and TGF-ß1 mRNA were given relative to expression of glyceraldehyde phosphate dehydrogenase mRNA in each individual sample.
Statistics
Values are presented as mean ± SEM or as percentages, unless otherwise indicated. The 
2 test or Fisher’s exact test was used to compare percentages. For parametric data, intergroups comparison was made by ANOVA and unpaired Student’s t test. Cumulative survival curves were constructed by the Kaplan-Meier method. P < 0.05 was considered significant.
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Results |
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Poor survival rates in TG mice
A cohort of TG (n = 148; 75 males and 73 females) and WT (n = 50; 25 males and 25 females) mice were monitored from 5–15 mo of age for cardiac function and survival. In both male and female TG groups, premature death started from 8 mo of age. Survival then progressively declined in both groups but remained higher for females throughout the observation period (78% vs. 41% at 12 mo and 56% vs. 12% at 15 mo; P < 0.001; Fig. 1
). All but two WT mice survived to 15 mo of age (Fig. 1).
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). Compared with the respective WT group, lung weight was 72% greater in TG males and 40% greater in TG females. At autopsy, a significantly higher incidence of pleural effusion, atrial thrombus, and lung congestion was found in TG vs. WT mice of the same gender (Table 1), indicating the presence of heart failure. All these events were more frequent in TG males than females.
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and 3. Heart rate was constantly and similarly higher in TG than in WT mice of both sexes throughout the study period, confirming the constitutive activation of ß2-adrenergic signaling (Fig. 2A). In WT mice, all echocardiographic parameters were largely unchanged over the study period and males had greater LV dimensions and LV mass than females consistent with their greater body mass (Figs. 2 and 3). At 6 mo, echocardiographic parameters were very similar between TG and WT groups. Between 9 and 15 mo, however, male and female TG mice showed a progressively enlarged LV cavity with the differences in LV dimensions between TG and WT mice being greater in males (Fig. 2A). Progressive LV dilatation was accompanied by reduction in wall thickness in TG mice during systole (data not shown) and diastole detected from 12 mo of age, again with more severe wall thinning in TG males (Fig. 2B). At 12 and 15 mo of age, male TG mice exhibited markedly increased LV mass compared with WT, indicating development of LV hypertrophy (Fig. 2A). In contrast, alterations in LV mass in TG females were minor relative to WT counterparts. The ratio of LVEDd/WTd, used to quantitate LV dilatation in relation to wall thickness, was markedly increased in TG males from 9–15 mo of age compared with TG females or WT males, evidence for dilated cardiomyopathy in male TG mice (Fig. 2C). TG males and females showed gradual decline in FS and WTI, which was more pronounced in TG males (Fig. 3, D and E). Representative 2-D and M-mode images from WT and TG mice (Fig. 3, A–C) illustrate such marked differences.
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, A–E). Cross-sectional area of myocytes was 2.7-fold greater in TG males and 1.8-fold greater in females compared with WT counterparts (Fig. 4H), and were larger in male than female TG mice. Collagen content was 5.5-fold greater in TG males and 3.4-fold greater in TG females compared with WT counterparts with more collagens in male than in female hearts (Fig. 4G). Such histological abnormalities were absent in WT hearts up to 15 mo of age, and there was no sex difference in myocyte size or collagen levels in WT mice.
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) remained infrequent, but the percentages were increased compared with 5-mo-old WT counterparts (both P < 0.05, Fig. 4I). In 15-mo-old TG males, the numbers of TUNEL-positive nuclei were greater than in the age-matched WT males and TG females (both P < 0.05, Fig. 4I), indicating an increased frequency of myocyte apoptosis in TG males.
Myocardial expression of -MHC and TGF-ß1
Expression of -MHC was significantly reduced by about 60% in the LV from 15-mo-old TG mice in comparison to WT mice (P < 0.01 for both genders, Fig. 5, A and C). This change was, however, similar between TG males and females. The LV from 15-mo-old TG males had elevated expression of TGF-ß1 compared with age-matched WT males (P < 0.01, Fig. 5, B and C). TGF-ß1 expression in the LV from 15-mo-old TG females was not significantly different from that in age-matched WT females.
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). Compared with intact females of both genotypes, ovariectomized females had a marked uterine atrophy (Table 2), a change known to be attributable to reduced levels of estrogen (14).
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). Unexpectedly, both TG female and male mice tended to survive better than nonoperated counterparts (Fig. 1), and this trend was more apparent in TG males (P = 0.071) than in females (P = 0.202).
Ovariectomy did not change heart weights significantly. Castration was associated with reduction in heart weights by 26% in WT and 39% in TG males (Table 1). Compared with the intact TG counterparts, TG males, but not females, had significant reductions in lung weight and incidence of pleural effusion, and a trend for a lower incidence of atrial thrombosis (Table 1), indicating less severe hypertrophy and left heart failure.
In male and female WT mice with gonadectomy, echocardiography over 6–15 mo of age showed no significant change in cardiac function and chamber size compared with intact WT mice (data not shown). This was also the case for TG females (Fig. 6). In contrast, gonadectomized TG males had a significant improvement in FS, partial reversal of LV dilatation, and reduction in LV mass (All P < 0.01, Fig. 6). The marked increase in LVEDd/WTd ratio, an indicator of dilated cardiomyopathy in intact TG males, was largely prevented (Fig. 6). In male and female TG mice, heart rate remained the same as in intact TG mice and was markedly higher than in WT mice with gonadectomy (P < 0.01, data not shown).
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-MHC and TGF-ß1 relative to WT counterparts were similar to that seen in the intact TG mice (preliminary data not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The development of cardiomyopathy in the ß2-AR TG lines requires a critical level of transgene overexpression (25). In the ß2-AR TG strain used, there was no significant difference in the ß2-AR density level between 6-mo-old male and female TG mice. At 15 mo of age, expression of endogenous -MHC was comparable in the heart of male and female TG mice, again indicating the lack of sex difference in the activity of the -MHC promoter that drives expression of the ß2-AR transgene. Moreover, the tachycardiac phenotype during the entire study period was similar among TG males and females with and without gonadectomy, suggesting that the degree of ß2-adrenergic stimulation to the heart was comparable. All of these indicate that the expression of ß2-AR transgene is not affected by gender or by gonadectomy.
Because the cardiomyopathy in this strain is characterized by loss of myocytes, ventricular dysfunction, and interstitial fibrosis, we elected to explore whether sex differences exist in myocyte apoptosis and in expression of -MHC and TGF-ß1. A role for myocyte loss in the development of cardiomyopathy and heart failure has been postulated by a number of studies in different species, including human, and myocyte apoptosis can be induced by activation of the stimulatory G protein-mediated signaling pathway (38). Further, sex differences in myocyte apoptosis have been reported (16, 36, 37). In TG males, a 2.7-fold increase in myocyte size and a 5.5-fold increase in fibrosis lead to only 20–30% increase in heart weight and LV mass. This implies substantial cell loss. While TUNEL-positive myocyte nuclei were more frequently seen in both 15-mo-old male and female TG than WT mice, the frequency was greater in TG males than females. Necrosis may also play a key role in myocyte loss in this model. Sustained high levels of heart rate and ventricular contractility in the ß2-AR TG mice could lead to a relative insufficiency of coronary blood supply and increased energy expenditure. Consequently, myocyte death due to necrosis is likely to occur in addition to that due to apoptosis. A recent study showed that, compared with female counterparts, the male ß2-AR TG heart is more susceptible to ischemic reperfusion injury with lower ATP levels in the myocardium (39). Indeed, we frequently observed in TG mice, particularly in TG males, ECG alterations similar to those seen in ischemic heart disease (data not shown).
Reduced expression of -MHC occurs in the hypertrophied and failing myocardium and is related to the attenuation of myocardial inotropy (34, 40). We observed a substantial down-regulation of -MHC expression in the LV of TG mice. However, no difference was observed between male and female TG mice, although TG females had a better preserved LV function. Ventricular expression of TGF-ß1 was elevated by 30% in 15-mo-old TG males but not in TG females. TGF-ß1 is known as a profibrotic, prohypertrophic, and proapoptotic cytokine (41, 42, 43, 44), and its enhanced expression may contribute to sex differences in the histological abnormalities.
Sex differences have recently been reported in several gene-targeted mouse strains with cardiomyopathy or hypertrophy due to expression of mutant -MHC (11) or phospholamban (8), overexpression of tumor necrosis factor- receptor (9) or 1B-AR (10), and disruption of natriuretic peptide receptor-A (12), FKBP12.6 (13) or peroxisome proliferator-activated receptor (7). In all these strains, with exception of the 1B-AR TG strain (10), male mice displayed more pronounced abnormalities in cardiac function and morphology than female counterparts. A survival advantage in females has also been observed in some strains (7, 8, 9, 12). In these strains, the more severe cardiac remodeling and dysfunction seen in males are also accompanied by a poorer rate of survival (8, 9, 10, 12). However, in contrast to the present study where the average age of cardiac deaths was similar in males and females, in most other strains the onset of death from cardiomyopathy started to occur at a younger age in males than females (8, 9, 10, 12). The mechanisms responsible for sex differences in cardiac phenotype in these various models are largely unknown.
A variety of human cardiovascular diseases show an increase in prevalence following menopause, suggesting the importance of prevailing sex hormone levels during adult life. We therefore elected to perform gonadectomy post weaning to examine the importance of prevailing levels of gonadal hormones in this model. Ovariectomy did not remove the survival advantage; indeed, the survival was somewhat improved compared with the nonoperated counterparts. This finding argues against a significant contribution from circulating estrogen to the survival benefit seen in female TG mice. Orchiectomy was associated with a strong tendency to a better survival and significant improvement in ventricular structure and function compared with intact TG males. Indeed, the sex differences in ventricular remodeling and dysfunction were diminished following orchiectomy. Taken together, these results lead to an intriguing suggestion that sex differences in the TG mice appear to be more dependent on adverse consequences of circulating testicular hormones rather than on a protection afforded by ovarian hormones. We believe that this is the first study the examine the role of gonadal status on cardiomyopathic phenotypes in gene-targeted male and female mice.
In conclusion, cardiomyopathy and heart failure induced by cardiac ß2-AR overexpression at a high level were significantly less severe in TG females than in males, and this was associated with a marked survival advantage. The mechanism for the observed sex differences does not appear to be due to a protective role of circulating female hormones but rather to an adverse consequence of testicular hormones.
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Footnotes |
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Abbreviations: ARs, Adrenergic receptors; 2-D, two-dimensional; dP/dtmax and dP/dtmin, rise and fall in LV pressure; FS, fractional shortening; LV, left ventricle (left ventricular); LVEDd, LV end-diastolic dimension; MHC, myosin heavy chain; mo, month(s); TG, transgenic; TUNEL, terminal transferase-mediated deoxyuridine triphosphate nick-end labeling; WT, wild-type; WTd, wall thickness at diastole; WTI, wall-thickening index.
Received December 31, 2002.
Accepted for publication May 12, 2003.
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References |
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-deficient mice. J Clin Invest 102:1083–1091[Medline]
1B-adrenergic receptor induces dilated cardiomyopathy. Am J Physiol 281:H931–H938
-MHC promoter is downregulated in hypertrophied and failing myocardium. Cardiovasc Res 47: 133–141
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