Testosterone Causes Decrease in the Content of Skeletal Muscle Myostatin

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1 Brief Paper : Physiology Testosterone Causes Decrease in the Content of Skeletal Muscle Myostatin Shigeo Kawada *, Makoto Okuno ** and Naokata Ishii ** * Department of Human and Engineered Environmental Studies, Graduate School of Frontier Sciences, The University of Tokyo Kashiwanoha, Kashiwa-shi, Chiba Prefecture, Japan kawada@k.u-tokyo.ac.jp ** Department of Life Sciences, Graduate School of Arts and Sciences, the University of Tokyo Komaba, Meguro-ku, Tokyo, Japan [Received December 21, 2005 ; Accepted April 25, 2006] Myostatin, a potent negative regulator of skeletal muscle growth, plays an important role in the regulation of muscle size. However, the mechanisms by which muscle myostatin production is regulated have not been fully understood. We hypothesized that testosterone, a positive regulator of muscle growth, causes a reduction of muscular myostatin content. To test this, we measured the content of myostatin in skeletal muscle after injection of testosterone at varied doses. Male C57BL/6j mice (age, 11 wk; n= 6 for each group) were subjected to subcutaneous injections of testosterone (0.3, 0.6, or 1.2mg/ day per 100g body weight) for 3 days. On the 4th day, the gastrocnemius/ plantaris complex (Gast/ Plant) and the soleus muscles were dissected and the content of myostatin protein was measured. Injection of testosterone tended to cause increases in body weight, Gast/ Plant and soleus muscle wet weight when compared to the control group. Myostatin content in the Gast/ Plant muscle decreased signifi cantly after the injection at 1.2mg per 100g body weight when compared to both control and after the injection at 0.3mg per 100g body weight. In the soleus muscle, the myostatin content also decreased signifi cantly after the injection at 0.3, 0.6 or 1.2mg per 100g body weight when compared to the control group. These results suggest that testosterone plays a part in the regulation of myostatin content in skeletal muscle. Keywords: myostatin, testosterone, negative regulator [] 1. Introduction Myostatin, a member of the TGF-ß superfamily, is a potent inhibitor of skeletal muscle growth [Lee and McPherron (2001)]. A myostatin-null human shows larger musculature and strength [Schuelke et al. (2004)]. It has also been demonstrated that the mutation of the myostatin gene causes hyperplasia in addition to hypertrophy of skeletal muscle fi bers [Lee and McPherron (2001)], whereas an increase in systemic myostatin causes skeletal muscle atrophy [Zimmers et al. (2002)]. Mechanical loading of skeletal muscle has been thought to be one of the critical factors in the regulation of myostatin production within muscle. The mouse hindlimb muscles respond to overloading with a decrease in myostatin protein content [Kawada et al. (2001)]. For human skeletal muscles, it has also been shown that heavy resistance training causes muscle hypertrophy associated with the reduction in myostatin expression [Roth et al. (2003)]. However, it remains unclear whether other factors including hormones and growth factors are involved in the regulation of myostatin production within skeletal muscle. Testosterone has long been regarded as one of the anabolic hormones for skeletal muscle, and its supplementation has been demonstrated to cause muscle hypertrophy [Bhasin et al. (2001)]. However, the effect of testosterone on myostatin production within skeletal muscle has not been fully understood, because no direct evidence has been put forward on muscle myostatin production by testosterone administration. Because the 5 -regulatory region of the myostatin gene has several binding sites for the androgen-receptor complex [Ma et al. (2001)], we hypothesized that testosterone down-regulates the transcription of myostatin, thereby causing the 44

2 Regulation of Myostatin reduction of skeletal muscle myostatin content. To test this hypothesis, the present study examined whether testosterone injection causes the reduction of myostatin content in mouse skeletal muscles. 2. Methods 2.1. Animals Male C57BL/6j mice (age, 11wk) were used. They were housed in an animal room with regulated temperature (22 ), humidity (60% ) and illumination cycles (12-h light and 12-h dark). They were allowed to eat commercial mouse chow (CLEA, Japan) and drink water ad libitum. Animal care and all experimental procedures employed were in accordance with the policy statement of the University of Tokyo on research with experimental animals. The study was approved by the Ethical Committee for Animal Experiments at the University of Tokyo Experimental design The mice were randomly assigned into control and experimental groups (n = 6 for control; n = 18 for experimental). It has been reported that testosterone injection of 0.6mg/ 100g body weight daily for one week causes a maximum muscle hypertrophy in impuberal rats [Eisenberg and Gordan (1950)]. Therefore, the mice were subjected to a subcutaneous injection of testosterone (Wako Co, Japan) at three different doses of 0.3, 0.6, and 1.2mg/ 100g body weight/ day for 3 days (n = 6 for each dose). It has been shown that hindlimb suspension of a mouse causes an increase in myostatin expression within one day, but the elevated expression gradually returns to the original level by the 7th day [Carlson et al. (1999)]. Also, our previous study has shown that myostatin responds to muscle reloading subsequent to unloading within a few days [Kawada et al. (2001)]. These studies suggest that changes in myostatin content take place within the initial few days of experiments. Therefore, the muscle specimens were dissected on the 4th day in the present study. The hormone was dissolved in sesame oil and the vehicle of 300µl was injected daily at the same time of the day. The control group was injected with the same volume of sesame oil. On the 4th day, the mice were euthanized by an over-dose intraperitoneal injection of sodium pentobarbital, and the gastrocnemius/ plantaris complex (Gast/ Plant) and soleus muscles were dissected from the hindlimb. The muscle samples were snap frozen in liquid nitrogen and stored at -80 until analysis Western blotting analysis Tissue samples were homogenized in a buffer containing 10-mM Tris-HCl (ph 7.4), 5% sodium dodecyl sulfate and 5% 2-mercaptoethanol. The homogenate was boiled for 3 minutes, then centrifuged at 13,000 g (4 ) for 15 minutes. The supernatant was removed and its protein concentration was determined using a protein determination kit (Bio-Rad, Richmond, CA). Then, equal amounts of extracted muscle proteins (100µ g total protein) were separated by sodium dodecyl sulfate-polyacrylamide (12%) gel electrophoresis (SDS-PAGE). Proteins on a gel were transferred onto poly vinilidene difl uoride (PVDF) membrane (ATTO, Japan) electrophoretically. The membrane was reacted with anti-myostatin antibody (1:250) and then with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG [1:2000 (American Qualex, CA)] as a secondary antibody. The primary antibody is a rabbit polyclonal antibody raised against amino acids of mouse myostatin origin [Kawada et al. (2001)]. Band density was determined by using the gel plotting program of the NIH Image (version 1.61; National Institutes of Health, Bethesda, MD). The band density was used as an indicator of myostatin content in the same amount of solubilized proteins. Its absolute value was then expressed relative to that for the control Statistical analysis The differences between groups were examined with one-way analysis of variance (ANOVA) followed by Games-Howell post-hoc test. P value less than 0.05 was considered as statistically signifi cant. 3. Results Testosterone injection appeared to cause slight increases in the body weight and the wet weight of both Gast/ Plant and soleus muscles when compared to the control group, but the changes were not 45

3 Table 1 Effects of testosterone injection on body and muscle weights. control experimental group Injection of testosterone (mg/100g body weight) mg 0.6mg 1.2mg body weight (g) pre-experiment 24.1 (0.95) 24.2 (0.52) 24.9 (1.26) 24.4 (0.97) post-experiment 24.9 (1.23) 25.7 (0.77) 26.0 (1.38) 26.3 (1.37) Increase in body weight (%) 3.37 (0.97) 6.42 (2.03) 4.40 (1.38) 7.93 (3.02) Muscle wet weight (mg) Gast/ Plant [post-experiment (mg)] (8.14) (3.43) (9.21) (6.02) Soleus [post-experiment (mg)] 7.55 (0.45) 7.72 (0.50) 8.52 (0.73) 8.40 (0.80) Means and S.D. (in brackets) are shown. A Relative myostatin content control 0.3mg 0.6mg 1.2mg Figure 1A Injection of testosterone (mg/100g body weight) B Relatine myostatin content * control 0.3mg 0.6mg 1.2mg Figure 1B Injection of testosterone (mg/100g body weight) Figure 1 Effects of testosterone injection on the myostatin content in Gast/Plant (A) and soleus (B). Bars denote S.E.M. *Signifi cantly different (P < 0.05) from values for control (vehicle injected). Signifi cantly different (P < 0.05) from values for 0.3mg-injected group. signifi cant (Table 1). Myostatin content in the Gast/ Plant muscle decreased signifi cantly after the injection of 1.2mg testosterone (per 100g body weight) as compared to both control and 0.3mg testosterone injected groups (Figure 1A). In the soleus muscle, the myostatin content showed signifi cant decreases after the injections of 0.3, 0.6 and 1.2mg testosterone when compared to the control group (Figure 1B). 4. Discussion Myostatin is known to be a potent negative regulator of skeletal muscle growth [Lee and McPherron (2001)]. It has been shown that myostatin stimulates the activity of p21, a cyclin-dependent kinase inhibitor, thereby suppressing the activity of Cdk2, an enzyme involved in the cell cycle progression [Thomas et al. (2000)]. However, the direct effect of myostatin on protein synthesis and/ or degradation within existing muscle fi bers has not been well understood, although a study has shown that myostatin binds to serine/threonine kinase receptors and activates subsequent phosporylation cascades [Lee and McPherron (2001)]. Several studies with experimental animals or humans have shown that skeletal muscles subjected to mechanical stress respond with a decrease in myostatin content and muscle hypertrophy [Kawada et al. (2001); Roth et al. (2003); Kim et al. (2005)]. In addition to mechanical stress, local circulatory change may infl uence the muscle myostatin content, because our previous study has shown that blood fl ow 46

4 Regulation of Myostatin restriction in hindlimb muscles causes a decrease in myostatin content and muscle hypertrophy [Kawada and Ishii (2005)]. However, additional factors, including systemic hormones and local growth factors, are also to be considered, because some of them are shown to be readily changed in response to exercise and mechanical loading [Psilander et al. (2003); Goto et al (2005)]. For instance, heavy resistance exercise causes an increase in serum testosterone level in male individuals [Ahtiainen et al. (2005)]. Testosterone may infl uence the myostatin production in muscle, because the 5 -regulatory region of the myostatin gene has several binding sites for the androgen-receptor complex [Ma et al. (2001)]. In the present study, testosterone injection caused a decrease in the content of myostatin in both Gast/ Plant and soleus muscles. Myostatin content in the Gast/ Plant muscle decreased signifi cantly after the injection of 1.2mg testosterone (per 100g body weight), whereas in the soleus muscle, the myostatin content showed signifi cant decreases after the injections of 0.3, 0.6 and 1.2mg testosterone when compared to the control group. It has been shown that both the androgen receptor and myostatin protein are more abundant in fast type muscles than in slow type muscles [Bircout et al. (1994); Carlson et al. (1999)]. This suggests that the sensitivity of myostatin response to testosterone differs with each muscle. Regarding the myostatin response to mechanical loading, our previous study has shown that the percentage reduction of myostatin content during a 2-day reloading after a 14-day unloading is larger in the soleus muscle (-36%) than in the Gast/ Plant muscle (-30%) in mice [Kawada et al. (2001)], suggesting that the myostatin response is more sensitive in slow type muscle than in fast type muscle. The present reduction in myostatin content after injection of testosterone may be mediated by the decreased synthesis and/ or increased breakdown of myostatin. Therefore, the present study did not conclude that the transcription of the myostatin gene is suppressed by the androgen-receptor complex although changes in myostatin protein are of more physiological signifi cance. Also, the present study failed to show signifi cant muscle hypertrophy in association with the decrease in myostatin. This was likely due to the short period (3 days) of testosterone treatment. Acknowledgement This study was supported by the grant-in-aid (No to N.I.) from the Japan Ministry of Education, Culture, Sciences, and Technology. References Ahtiainen JP, Pakarinen A, Alen M, Kraemer WJ, Hakkinen K (2005) Short vs. long rest period between the sets in hypertrophic resistance training: infl uence on muscle strength, size, and hormonal adaptations in trained men. 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5 Name: Shigeo KAWADA Affi liation: Department of Human and Engineered Environmental Studies, Graduate School of Frontier Sciences, The University of Tokyo Address: Kashiwanoha, Kashiwa-shi, Chiba Prefecture, Japan Brief Biographical History: Kitasato Institute for Life Sciences, Kitasato University (Post-doctoral fellow) Department of Human and Engineered Environmental Studies, Graduate School of Frontier Sciences, The University of Tokyo (Associate researcher) Main Works: Skeletal muscle hypertrophy after chronic restriction of venous blood fl ow in rats. Med Sci Sports Exerc. 2005,37(7) Membership in Learned Societies: Japanese Society of Physical Fitness and Sports Medicine American Physiological Society National Strength & Conditioning Association 48