The effects of myokines on osteoclasts and osteoblasts

https://doi.org/10.1016/j.bbrc.2019.07.127Get rights and content

Highlights

  • Myotube conditioned media (CM) inhibited osteoclastogenesis and bone resorption.

  • Myotube CM increased preosteoblast viability and migration.

  • Systemic treatment with myotube CM increased bone mass in mice.

  • Muscle may play beneficial roles in bone health via secretion of anabolic factors.

Abstract

Recently, muscle has received much attention as an endocrine organ regulating other biological targets, including the pancreas, liver, and adipose tissue. Although there is a possibility that muscle-secreting factors biochemically affect bone metabolism in a paracrine manner, the net effects of myokines on the biology of osteoclasts and osteoblasts, particularly on bone mass in vivo, have not yet been thoroughly investigated. Therefore, we performed in vitro as well as animal experiments using conditioned media (CM) collected from C2C12 myoblast and myotube cultures to better understand the interactions between muscle and bone. Compared with non-CM (i.e., control) and myoblast CM, myotube CM markedly inhibited in vitro bone resorption through the suppression of osteoclast differentiation and resorptive activity of individual osteoclasts. Consistently, the expressions of osteoclast differentiation markers, such as tartrate-resistant acid phosphatase (Trap) and calcitonin receptor (Ctr), decreased with myotube CM. Myotube CM significantly stimulated preosteoblast viability and migration and reduced apoptosis, thereby resulting in an increase in calvaria bone formation. Importantly, systemic treatment with myotube CM for 4 weeks increased bone per tissue volume by 30.7% and 19.6% compared with control and myoblast CM, respectively. These results support the hypothesis that muscle plays beneficial roles in bone health via secretion of anabolic factors, in addition to mechanical stimuli, and importantly indicate that muscle-derived factors can be potential therapeutic targets against metabolic bone diseases.

Introduction

Muscle and bone are the largest tissues of non-obese humans and share the common purposes of protecting internal organs and enabling movement [1]. Interestingly, clinical observations indicate that changes in muscle and bone health are strongly correlated throughout an individual's lifetime [2,3]. For example, muscle atrophy and bone loss are often present in individuals of similar age groups [4,5]. Because both sarcopenia and osteoporosis are well-known risk factors for fragility fractures [6], a better understanding about muscle–bone interactions underlying the parallel changes is essential for effective prevention of fractures.

Although interactions between muscle and bone can be bi-directional, their highly integrated nature has been conventionally explained by muscle force-generated mechanical signals, which transduce anabolic activity in the adjacent bone [7,8]. Importantly, muscle has recently received much attention as an endocrine organ that regulates other biological targets, including the pancreas, liver, and adipose tissue [9]. Therefore, besides the mechanostat theory, there is a possibility that muscle-secreting factors—defined as “myokines”—biochemically affect bone metabolism in a paracrine manner [10,11]. When open tibial fractures were covered with muscle flap in a murine model, the rate and quality of bone repair were markedly improved compared with fractures covered with equally vascularized fasciocutaneous tissue [12,13], which supports the aforementioned hypothesis. However, despite this plausibility, the net effects of myokines on the biology of osteoclasts and osteoblasts, particularly on bone mass in vivo, have not yet been thoroughly investigated. With an aim of further understanding how muscle interacts with bone, we performed in vitro as well as animal experiments using conditioned media (CM) from C2C12 myoblast and myotube cultures.

Section snippets

Collection of C2C12 myoblast and myotube CM

Mouse C2C12 myoblasts were purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained in DMEM supplemented with 10% FBS, 20 mM HEPES, 2 mM l-glutamine, and antibiotics (Life Technologies Corp., Carlsbad, CA) at 37 °C in a humidified atmosphere comprising 5% CO2. CM was collected after further incubation for 24 h in serum- and phenol red-free media, and these were regarded as myoblast CM. To induce myogenic differentiation, cells were grown to 90% confluency in

Suppression of osteoclastogenesis and in vitro bone resorption using myotube CM

The effects of myotube CM on the biology of osteoclasts were compared with those of myoblast CM and non-CM (control). When primary mouse BMMs were cultured with various CM concentrations in the presence of M-CSF and RANKL, 10%, 25%, and 50% myotube CM significantly inhibited osteoclast differentiation, as shown by the results of TRAP staining (Fig. 1A). Because the effect on osteoclastogenesis was already at a maximum with 25% myotube CM, subsequent experiments on osteoclasts were performed

Discussion

Since Pederson et al. [18] had first coined the term “myokines” to describe cytokines or other peptides that are produced and secreted by muscle fibers, several lines of evidence have come to support the role of muscle as an endocrine organ [9,10]. In addition, in the present study, we have demonstrated the distinct effects of myotube versus myoblast CM on bone cells. Myotube CM inhibited in vitro bone resorption through the suppression of osteoclastogenesis and osteoclastic resorptive

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) [grant numbers 2016M3A9E8941329 and 2019R1A2C2006527].

References (23)

  • S. Verschueren et al.

    Sarcopenia and its relationship with bone mineral density in middle-aged and elderly European men

    Osteoporos. Int.

    (2013)
  • Cited by (8)

    • Muscle-derived extracellular vesicles improve disuse-induced osteoporosis by rebalancing bone formation and bone resorption

      2023, Acta Biomaterialia
      Citation Excerpt :

      The osteoblasts were passaged by trypsinization for further experiments. Osteoclast formation was carried out as previously described with slight modification [32]. Briefly, primary bone marrow cells were collected by flushing the femurs and tibias of six-week-old male C57BL/6J mice.

    • Effects of Muscles on Bone Metabolism—with a Focus on Myokines

      2022, Annals of Geriatric Medicine and Research
    View all citing articles on Scopus
    1

    Jin Young Lee and So Jeong Park contributed equally to this work.

    View full text