Estrogens Maintain Skeletal Muscle and Satellite Cell Functions
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Y KITAJIMA and Y ONO Estrogens regulate muscle 229:3 267–275 Research functions Estrogens maintain skeletal muscle and satellite cell functions Correspondence Yuriko Kitajima and Yusuke Ono should be addressed to Y Ono Department of Stem Cell Biology, Atomic Bomb Disease Institute, Nagasaki University Graduate School Email of Biomedical Sciences, Nagasaki, Japan [email protected] Abstract Estrogens have crucial roles in an extensive range of physiological functions regulating Key Words cellular proliferation and differentiation, development, homeostasis, and metabolism. f estrogens Therefore, prolonged estrogen insufficiency influences various types of tissues f skeletal muscle expressing estrogen receptors (ERs). Although ERs are expressed in skeletal muscle f muscle atrophy and its stem cells, called satellite cells, how prolonged estrogen insufficiency affects f satellite cells their function remains unclear. In this study, we investigated the effect of estrogen f females reduction on muscle in young ovariectomized (OVX) female mice. We found that reduced estrogens resulted in muscle atrophy in a time-dependent manner. Muscle force generation was reduced in OVX mice. Interestingly, prolonged estrogen insufficiency Endocrinology shifted fiber types toward faster myosin heavy chain isoforms. The number of satellite of cells per isolated myofiber was unchanged, while satellite cell expansion, differentiation, and self-renewal were all markedly impaired in OVX mice. Indeed, muscle regeneration was significantly compromised in OVX mice. Taken together, our results demonstrate Journal that estrogens are essential for comprehensively maintaining muscle function with its Journal of Endocrinology insufficiency affecting muscle strength and regeneration in young female mice. (2016) 229, 267–275 Introduction Estrogens have crucial roles in an extensive range of mineral bone density by inhibition of bone remodeling physiological functions regulating cellular proliferation and resorption as well as increasing bone formation. and differentiation, development, homeostasis, and Indeed, prolonged estrogen insufficiency leads to loss of metabolism (Mauvais-Jarvis 2011). Estrogen insufficiency bone mineral density, resulting in fragility fractures. For is caused by a variety of factors such as menopause, example, a cross-sectional study showed that menstrual surgical ovariectomy, and chemotherapy. In females, dysfunction may be a possible risk factor contributing to insufficient energy intake leads to body weight loss that stress fractures in female endurance athletes (Duckham also causes hypoestrogenic menstrual abnormalities. The et al. 2012). Furthermore, a prospective study reported that female athlete triad, low energy availability, menstrual teenage female athletes with menstrual dysfunction had dysfunction, and low bone mineral density, is prevalent a greater risk of musculoskeletal injury (Rauh et al. 2010). in sports activities, particularly endurance sports such as Accumulating evidence suggests sex-specific differences marathons as well as aesthetic sports such as gymnastics and in muscle homoeostasis. Although the effects of androgens ballet (Matzkin et al. 2015). ERs are expressed in a variety of on muscle function have been evaluated extensively, the tissues including bone and muscle (Mauvais-Jarvis 2011). roles of estrogens in muscle have been largely unknown. Estrogens play an important role in the maintenance of Recent studies have revealed that the levels of estrogens http://joe.endocrinology-journals.org 2016 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JOE-15-0476 Printed in Great Britain Downloaded from Bioscientifica.com at 09/27/2021 05:18:36AM via free access 10.1530/JOE-15-0476 Research Y KITAJIMA and Y ONO Estrogens regulate muscle 229:3 268 functions are associated with muscle mass (McClung et al. 2006, (MyHC) (SC-71) and mouse anti-type IIb MyHC (BF-F3) Sitnick et al. 2006, Sugiura et al. 2006, Pollanen et al. 2011). antibodies were purchased from Deutsche Sammlung Ovariectomized (OVX) mice fail to fully recover muscle von Mikroorganismen (Braunschweig, Germany). A rat mass after hindlimb suspension-induced muscle atrophy anti-laminin α2 antibody was obtained from Alexis (San followed by reloading (McClung et al. 2006). This failure Diego, CA, USA). A goat anti-collagen type 1 antibody was to recover muscle mass in OVX mice may be associated purchased from Southern Biotech (Birmingham, AL, USA). with reduced p70S6K signaling (McClung et al. 2006, Slow-release 17β-estradiol pellets (0.01 mg/60 days) were Sitnick et al. 2006). Moreover, treatment with estrogens purchased from Innovative Research of America (Sarasota, attenuates immobilization-induced muscle atrophy, even FL, USA). Mounting medium containing DAPI for nuclear in male rats (Sugiura et al. 2006). These findings indicate staining and an M.O.M. kit were obtained from Vector that estrogens have a role in alleviating disuse-induced Laboratories (Burlingame, CA, USA). muscle atrophy and promoting regrowth after reloading. However, the effect of prolonged estrogen insufficiency on Animals muscle remains to be fully understood. Muscle tissue stem cells, called satellite cells, play Animal experimentation was approved by the crucial roles in providing myonuclei for postnatal Experimental Animal Care and Use Committee of the muscle growth as well as in muscle maintenance, repair, Nagasaki University, Nagasaki, Japan. Six-week-old regeneration, and hypertrophy in adults (Dumont et al. female C57BL/6 mice (Charles River Laboratories) were 2015). Thus, understanding the regulation of satellite ovariectomized under anesthesia. The slow-release cells is critical to elucidate the mechanisms in skeletal 17β-estradiol pellets were implanted at the same time as muscle maintenance. Several studies have reported that OVX for estrogen replacement as described previously estrogens appear to favor muscle regeneration after injury (Kitajima et al. 2015). Sham-operated mice were used as (Diel 2014). Estrogens attenuate exercise-induced muscle a control. All mice were killed at 2, 4, 8, and 24 weeks damage by downregulation of inflammatory responses after surgery. Endocrinology (Tiidus et al. 2001), increase the number of satellite cells, of and stimulate satellite cell proliferation after running exercise (Enns & Tiidus 2008). However, whether Grip test extrinsic factors influence satellite cell activity or the Journal Maximal limb muscle force was measured by a Grip intrinsic potential of satellite cells is affected by estrogen Strength Meter (Columbus Instruments, Columbus, insufficiency remains unclear. OH, USA). Three sets of ten successive measurements In this study, we explored the impact of prolonged were carried out to assess forelimb grip strength. The estrogen reduction on muscle and satellite cells in young maximum values in three sets of experiments were used OVX female mice. Estrogen insufficiency resulted in muscle for data analysis. atrophy, reduced muscle force generation, and a shift to a faster fiber-type distribution. Although the number of satellite cells per isolated single myofiber was unaltered, Muscle regeneration population expansion, myogenic differentiation, and To induce muscle injury, 50 μL of 10 μM cardiotoxin (CTX, self-renewal of satellite cells were significantly reduced Sigma-Aldrich) was injected intramuscularly into the tibialis in OVX mice. Indeed, muscle regeneration was impaired anterior (TA) muscle of anesthetized mice using a 29 G by a reduction of estrogen levels. Therefore, these results 1/2 insulin syringe. Regenerating muscles were isolated at indicate that estrogens are crucial to maintain the functions 14 days after CTX injection. Transverse sections of the of both muscle and satellite cells in young female mice. muscles were prepared using a cryostat and immunostained. Materials and methods Satellite cell isolation and culture Extensor digitorum longus (EDL) muscles were isolated Antibodies and reagents and then digested by type I collagenase as described Antibodies and reagents were obtained from the following previously (Collins & Zammit 2009). Satellite cells were sources: mouse anti-type IIa myosin heavy chain obtained from the isolated myofibers and cultured in a http://joe.endocrinology-journals.org 2016 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JOE-15-0476 Printed in Great Britain Downloaded from Bioscientifica.com at 09/27/2021 05:18:36AM via free access Research Y KITAJIMA and Y ONO Estrogens regulate muscle 229:3 269 functions mitogen-rich medium (GlutaMax DMEM supplemented We found an atrophic uterus in OVX mice and a gradual with 10% horse serum, 0.5% chicken embryo extract increase in the body weight of OVX mice compared with (CEE, US Biologica, Salem, MA, USA), and 1% penicillin– control mice as described previously (Fig. 1B, C and D) streptomycin) at 37°C with 5% CO2. (Kitajima et al. 2015). As a decrease in uterine size and increase in body weight are typical phenotypes of an estrogen- deficient state (Jansson et al. 2006), our ovariectomy-induced Immunostaining estrogen-deficient model was established successfully. Immunocytochemical analysis of satellite cells associated with myofibers was performed as described previously Estrogen insufficiency results in muscle atrophy and (Masuda et al. 2015). Samples were incubated with decreased muscle strength primary antibodies at 4°C overnight following blocking/ permeabilization with phosphate-buffered