Androgen Receptor Gene Knockout Male Mice Exhibit Impaired Cardiac Growth and Exacerbation of Angiotensin II-induced Cardiac Fibrosis

【摘要】  Androgen has anabolic effects on cardiac myocytes and has been shown to enhance left ventricular enlargement and function. However, the physiological and patho-physiological roles of androgen in cardiac growth and cardiac stress-induced remodeling remains unclear. We aimed to clarify whether the androgen-nuclear androgen receptor (AR) system contributes to the cardiac growth and angiotensin II (Ang II)-stimulated cardiac remodeling by using systemic AR-null male mice. AR knock-out (ARKO) male mice, at 25 weeks of age, and age-matched wild-type (WT) male mice were treated with or without Ang II stimulation (2.0 mg/kg/day) for 2 weeks. ARKO mice with or without Ang II stimulation showed a significant reduction in the heart-to-body weight ratio compared with those of WT mice. In addition, echocardiographic analysis demonstrated impairments of both the concentric hypertrophic response and left ventricular function in Ang II-stimulated ARKO mice. Western blot analysis of the myocardium revealed that activation of extracellular signal-regulated kinases (ERK) 1/2 and ERK5 by Ang II stimulation were lower in ARKO mice than those of WT mice. Ang II stimulation caused more prominent cardiac fibrosis in ARKO mice than in WT mice with enhanced expression of types I and III collagen and transforming growth factor-1 genes and with increased Smad2 activation. These results suggest that, in male mice, the androgen-AR system participates in normal cardiac growth and modulates cardiac adaptive hypertrophy and fibrosis during the process of cardiac remodeling under hypertrophic stress.

【关键词】  Androgen Receptor Knockout Impaired Exacerbation Angiotensin IIinduced Fibrosis

INTRODUCTION

Androgen exerts a variety of biological effects in many target organs, including male genitalia, brain, and skeletal tissues (1, 2). Most of these actions are mediated through the transcriptional control of particular sets of target genes by the nuclear androgen receptor (AR).1 AR is a ligand-inducible transcription factor that belongs to the nuclear receptor superfamily (3, 4). Upon hormone binding, AR is translocated from the cytosol into the nucleus where it binds specific promoter elements. A number of coregulators and/or coregulator complexes are then recruited to AR, which then activates or represses the transcription of various target genes (2, 3, 5-7). To clarify the physiological function of androgen via AR transcriptional regulation, we generated AR knock-out (ARKO) mice by means of the Cre-loxP system and have reported that ARKO male mice manifest late-onset obesity (8), high turnover osteopenia (9), and impaired brain masculinization (10).

In addition to the classic target tissues of androgens, the AR gene is also expressed in mammalian cardiomyocytes, suggesting that androgens may play a role in the heart (11). In fact, Marsh and colleagues (11) have shown that androgens produce cardiac hypertrophy by a direct, receptor-specific mechanism. They also revealed that androgens regulate functional expression of an L-type calcium channel in isolated rat ventricular myocytes, leading to a modulation of cardiac performance in males (12). Li et al. (13) reported that either castration or flutamide, an AR antagonist, markedly attenuated cardiac hypertrophy and fibrosis in guanylyl cyclase-A knock-out male mice. These previous studies have helped in delineating the influence of the androgen-AR system in the heart. However, to prove the physiological and pathophysiological roles of the androgen-AR system on cardiac structure and function, in vitro experiments as well as in vivo studies using pharmacological manipulations have limitations.

In the present study, to define the biological significance of cardiac AR function, we carried out studies using ARKO male mice. We obtained in vivo evidence that the androgen-AR system plays a pivotal role in normal cardiac growth and in cardiac protection against angiotensin II (Ang II)-induced cardiac fibrosis in male mice.

EXPERIMENTAL PROCEDURES

Animal Preparation-ARKO male mice were generated by targeted disruption of the AR gene using the Cre-loxP system. ARKO male mice with C57BL6/CBA hybrid backgrounds were generated and maintained as previously reported (8-10). In ARKO male mice, testicular androgen production seemed to be severely impaired, leading to a reduction in serum gonadal androgen levels, whereas serum adrenal androgen and estrogen levels remained normal (9). We used littermate wild-type (WT) male mice and ARKO male mice in this study and divided these mice into four groups; WT male mice with or without Ang II stimulation and ARKO male mice with or without Ang II stimulation. Ang II (WAKO, Japan) dissolved in saline was continuously and subcutaneously infused at a rate of 2.0 mg/kg/day for 2 weeks using an osmotic minipump (Alzet model 1002, Alza Corp., Mountain View, CA). Experiments were conducted in these male mice at 25 weeks of age. All experimental procedures were performed in accordance with the guidelines of the Animal Research Committee, The University of Tokushima Graduate School.