Ang II-stimulated Cardiac Dysfunction in ARKO Male Mice-We also evaluated the cardiac performance of these mice using echocardiography. Although left ventricular FS, a marker of systolic function, was not significantly different between WT and ARKO mice, Ang II stimulation significantly attenuated LV systolic function in ARKO mice compared with WT mice (Fig. 2).

Ang II-stimulated Cardiac ERK1/2 and ERK5 Activations Were Decreased in ARKO Male Mice-Because MAP kinase pathways have been shown to be involved in cardiac hypertrophy (18, 19) and function (20), we examined effects of Ang II stimulation on ERK1/2 and ERK5 activities. Ang II stimulation caused a significant increase in cardiac ERK1/2 and ERK5 phosphorylation in WT mice, whereas Ang II stimulation did not significantly enhance MAP kinase phosphorylation in ARKO mice (Fig. 3). Phosphorylation of other MAP kinases, c-Jun NH2-terminal kinase and p38, by Ang II were not observed in either WT or ARKO mice (data not shown). These results suggest that the blunted response to Ang II stimulation in ERK1/2 and ERK5 activities may be at least in part related to the aberrant cardiac hypertrophic response and cardiac dysfunction in ARKO mice.

FIG. 2.

Upper panel, representative echocardiogram of LV wall motion in WT and ARKO male mice with or without Ang II stimulation. Lower panel, echocardiographic measurements of LVDd, LVM/BW, RWT, and FS in WT and ARKO male mice with or without Ang II stimulation. LVDd, left ventricular diastolic dimension; LVM, left ventricular mass; BW, body weight; RWT, relative wall thickness; FS, fractional shortening. Values are expressed as mean ± S.E. *, p n = 25.

Aberrant Gene Expression of Cardiac Remodeling Factors in ARKO Male Mice-As shown in Fig. 4, the basal gene expression levels of BNP, MHCs, and types I and III collagen were not different between WT and ARKO mice. The ANP mRNA level was slightly lower in ARKO mice than in WT mice. Although Ang II stimulation markedly enhanced the expression levels of ANP and BNP mRNA in both WT and ARKO mice, the expression level of ANP mRNA was increased more in ARKO mice than in WT mice. Ang II stimulation did not affect MHC mRNA levels in either WT or ARKO mice, but enhanced MHC mRNA expression only in WT mice. Ang II stimulation enhanced both type I and III collagen mRNA expression levels in WT and ARKO mice, but the enhancement was more pronounced in ARKO mice. Although there were no significant differences in the AT1aR and AT2R mRNA levels between WT and ARKO mice with or without Ang II stimulation, ARKO mice showed a tendency for higher expression in these mRNA levels than WT mice in the presence or absence of Ang II stimulation (Fig. 5).

TGF-1 Expression and Smad Pathway Were Activated in ARKO Mice by Ang II Stimulation-TGF-1 has been identified as a contributor of cardiac fibrosis (21), and Ang II is known to affect TGF-1 expression (22). Upon binding of TGF-1 to its receptor, phosphorylation of Smad2 and its subsequent translocation to the nucleus is a critical step in TGF-1 signaling pathway (23, 24). Therefore, we examined the effects of Ang II on TGF-1 expression and Smad2 activation. Although Ang II stimulation significantly enhanced cardiac TGF-1 mRNA levels in both WT and ARKO mice, the degree of enhancement of its gene expression was more augmented in ARKO mice than in WT mice (Fig. 5). Consistent with the increase in TGF-1 mRNA expression, Ang II stimulation also caused a significant increase in cardiac Smad2 phosphorylation in both WT and ARKO mice with more Smad2 phosphorylation in ARKO than in WT mice (Fig. 6).

FIG. 3.

Effects of AR inactivation on cardiac ERK1/2 and ERK5 phosphorylation in WT and ARKO male mice with or without Ang II stimulation. ERK1/2 and ERK5 phosphorylation of the hearts were measured by Western blot analysis as described under "Experimental Procedures." The upper panels show the representative blots of phosphorylated forms of ERK1/2 and ERK5, and the middle panels show the blots of total (phosphorylated and unphosphorylated) ERK1/2 and ERK5, respectively. Lower panels show the densitometric analysis of phosphorylated ERK1/2 and ERK5 of WT (white bars) and ARKO (black bars) male mice. Values were normalized by arbitrarily setting the densitometry of control (without Ang II in WT male mice) to 1.0. Values are expressed as mean ± S.E. *, p n = 18.

DISCUSSION

Increased cardiac mass, used synonymously with cardiac hypertrophy, is one of the important cardiovascular risk factors (25). Although appropriate cardiac hypertrophy is an adaptive response to several forms of heart disease, excessive cardiac hypertrophy causes pathological cardiac remodeling. Cardiac mass increases at much greater rates in males than in females from puberty and throughout life, even after allometric adjustment for gender differences in overall body size (26, 27). The gender difference of LV mass has led to the hypothesis that sex hormones, such as androgen and estrogen, influence LV mass (25). In fact, estrogen has a preventive effect on cardiac hypertrophy (28-30), whereas androgen causes cardiac hypertrophy (13, 25, 28-30). In experimental animals, cardiac hypertrophy is suppressed by castration (13, 30, 31) and anti-androgens (32). Serum levels of testosterone, dehydroepiandrosterone, and its sulfate conjugate dehydroepiandrosterone sulfate decrease in males with chronic heart failure (33-36), and administration of testosterone results in an improvement of cardiac function in male patients with congestive heart failure (37). However, it is still unclear whether androgen plays a beneficial role in the regulation of cardiac structure and function.

To clarify the physiological roles of androgen on cardiac structure and function, we used ARKO male mice established using the Cre-loxP system, which allows the AR-null mutation to be passed on to offspring (8, 9). The present study demonstrated that cardiac volume and wall thickness were smaller in ARKO mice than those in WT mice without any difference in blood pressure or heart rate between these mice. The physiological and pathophysiological significance of androgen in male cardiac morphology and function was also examined. Our results indicate that androgen action is required for sufficient cardiac growth with adequate myofiber size, regardless of the presence or absence of cardiac stress in male mice.

Experimental and clinical observations indicate that Ang II stimulation induces hypertension (38, 39), pathological cardiac remodeling (40, 41), and heart failure (42, 43). When Ang II stimulation causes pressure or volume overload of the heart, the resulting cardiac hypertrophy is initially a compensatory response to preserve cardiac performance against cardiac load. It is also postulated that the concentric geometric remodeling with a reduction in the LV chamber size relative to wall thickness is an adaptation to preserve the LV pump function (44). The present study demonstrated that Ang II-stimulated ARKO male mice exhibit an aberrant cardiac hypertrophic response of the LV wall. Therefore, our results indicate that AR is required for the physiological hypertrophy of normal postnatal cardiac development in male mice, and for adaptive responses to cardiac stress such as Ang II stimulation.