FIG. 4.

Aberrant gene expression patterns of cardiac remodeling factors in ARKO male mice with or without Ang II stimulation. The left panel demonstrates the representative Northern blots of cardiac remodeling factors in WT and ARKO male mice with or without Ang II stimulation. The right panel shows the results of densitometric analyses of each parameter in WT (white bars) and ARKO (black bars) male mice. Values were normalized by arbitrarily setting the densitometry of control (WT male mice without Ang II) to 1.0. Values are expressed as mean ± S.E. *, p **, p n = 18.

FIG. 5.

Effects of AR deficiency on the gene expression of AT1aR (left), AT2R (middle), and TGF-1(right) with or without Ang II stimulation. The real-time PCR analyses of each parameter were performed in WT (white bars) and ARKO (black bars) male mice. Values were normalized by arbitrarily setting the measurement of control (WT male mice without Ang II) to 1.0. Values are expressed as mean ± S.E. *, p n = 12.

FIG. 6.

Cardiac Smad2 phosphorylation in WT and ARKO male mice with or without Ang II stimulation. Phosphorylated Smad2 levels of the heart were measured by Western blot analysis as described under "Experimental Procedures." The left panels show the representative blots of phosphorylated forms of Smad2, and the blots of total (phosphorylated and unphosphorylated) Smad2. The right panel shows the results of densitometric analysis concerning phosphorylated Smad2 in WT (white bars) and ARKO (black bars) male mice. Values were normalized by arbitrarily setting the densitometry of control (WT male mice without Ang II) to 1.0. Values are expressed as mean ± S.E., *p n = 6.

A critical role for ERK1/2 and ERK5 has been demonstrated in transgenic mice in the development of cardiac hypertrophy (18, 19). In the present study, we found that ERK1/2 and ERK5 were activated by Ang II stimulation in WT male mice, whereas their activities were lower in ARKO male mice after Ang II stimulation. These results are consistent with the macro- and microscopic observations of WT and ARKO male mice hearts, and suggest that ERK1/2 and/or ERK5 activation might be involved in Ang II-induced cardiac hypertrophy. It has been reported that Ang II infusion causes cardiac hypertrophy in rats with a concomitant increase in ERK1/2 activity (45). In addition, ERK5 activation has been shown to cause hypertrophy of cardiomyocytes (46) as well as the inhibition of endothelial cell death (47). Thus, the blunted ERK1/2 and ERK5 activation may be the mechanism whereby ARKO male mice exhibit reduced cardiac size with aberrant hypertrophic response to Ang II stimulation. However, further studies are needed to clarify the precise role of MAP kinases in AR-mediated intracellular signaling in the heart.

The present study demonstrated that the mRNA level of ANP in ARKO mice was reduced compared with WT mice. This result is consistent with the evidence that androgens increase ANP secretion via an AR-mediated mechanism in cultured rat myocytes (11). However, Ang II stimulation caused prominent enhancement of ANP gene expression in ARKO mice more than WT mice. If androgen actions were lacking under cardiac stress, it may be required that some ANP enhancers, including mechanical stress, strongly promote the ANP expression for supporting the cardiac performance. We also found the reduction of MHC gene expression in Ang II-stimulated ARKO mice compared with WT mice with Ang II loading. An up-regulation of the MHC gene relative to the MHC gene is observed in clinical conditions such as cardiac hypertrophy, cardiomyopathy, and congestive heart failure (48, 49). Because this up-regulation of the gene has been recognized as a compensatory response during cardiac remodeling, we speculate that aberrant MHC gene expression in ARKO mice is associated with impairment of adaptive cardiac hypertrophy under the condition of hypertrophic stress. Further investigation is needed to clarify this issue. And we found tendencies of higher AT1aR and AT2R gene expression in ARKO mice compared with WT mice, however, there were no statistical differences among those groups. Thus, we could conclude at least in this situation that AT1aR or AT2R expression levels are not a major cause of impaired cardiac hypertrophic response after Ang II stimulation in ARKO mice.

In addition to insufficient cardiac growth and aberrant hypertrophic responses, the present study demonstrated an enhanced cardiac fibrosis with up-regulation of collagen I and III gene expression in ARKO male mice after Ang II infusion. It has been reported that Ang II-induced proliferation of cardiac fibroblasts and an increase of collagen deposition contribute to an increase in cardiac muscle stiffness and the development of diastolic and systolic dysfunctions (50, 51). In fact, the present echocardiographic examination showed that the significant impairment of systolic function was observed in only ARKO male mice with Ang II stimulation. We also found that Ang II stimulation enhanced cardiac TGF-1 and Smad2 phosphorylation in ARKO mice more than in WT mice. TGF-1 is a powerful stimulator of the production of fibrillar collagens and other extracellular matrix components in a variety of cell types (52). A major signaling pathway after TGF-1 receptor activation is phosphorylation of Smad2/3 pathway (24). Hao et al. (53) showed that the levels of cardiac Smad2 are up-regulated after myocardial infarctions and that phosphorylation of Smad2 is attenuated by an Ang II type 1 receptor blockade. In addition, Kyprianou et al. (54, 55) revealed that endogenous androgens suppress the TGF- expression and Smad2/3 activation in the prostates of rats. They also showed that castration causes robust expression of TGF- and activation of Smad2/3 in the prostate (54, 55). Those previous reports and our results suggest that androgens have a protective role against AngII-stimulated cardiac fibrosis via suppression of TGF-1-Smad signaling. Taken together, the present observations provide novel in vivo evidence that the androgen-AR system plays a protective role against Ang II-induced aberrant cardiac remodeling that leads to systolic dysfunction.

ACKNOWLEDGMENTS

We thank Kazue Ishikawa for her technical help.