Understanding the mechanical loading-independent communication from skeletal muscle to bone: the role of muscle-derived exosomal miR-206 in regulating bone formation during muscle atrophy

The mechanical loading of skeletal muscle to bone is essential for bone development and remodeling. Besides the mechanical loading-dependent paradigm, the skeletal muscle also serves as an endocrine organ capable of secreting hormones to modulate bone metabolism. Recently, growing evidences indicate that myocytes can release muscle-specific microRNA-containing exosomes. In addition, it has been reported that the muscle-specific microRNAs (myomiRs) could be found in the circulation of the chronic obstructive pulmonary disease (COPD) and Duchenne muscular dystrophy (DMD) patients. Moreover, a series of myomiRs, including miR-1, miR-133a, miR-206 and miR-499 could inhibit osteoblastic activity in vitro. The recently published evidences have demonstrated that circulating microRNAs are stable in body fluids (e.g. serum) and transported within extracellular vesicles (e.g. exosome), indicating their potential role as extracellular genetic molecules for cell-cell communication. Thus far, studies addressing the role of muscle-derived exosomal myomiRs in regulating bone formation have not been reported.

In our preliminary studies, the applicants have profiled the exosome-containing microRNAs in the supernatant of the cultured C2C12 cells during microgravity simulation (MGS-C2C12) and identified miR-206 as the most dominant microRNA in these exosomes (Figure 1a-b). The applicants found that the higher exosomal miR-206 level in skeletal muscle and serum associated with the lower mRNA levels of bone formation marker genes and the smaller skeletal muscle volume in fractured patients with long- term bed rest (Figure 1c-e). Moreover, the applicants found increased exosomal miR-206 levels in skeletal muscle cells, serum and osteoblasts, but no changes in pri-miR-206 and pre-miR-206 levels within osteoblasts from Sprague-Dawley (SD) rats during unloading. Interestingly, the decreased bone formation rate (BFR/BS) and mineral apposition rate (MAR) in bone samples associated with the increased exosomal miR-206 in muscle cells and osteoblasts were observed during the last four weeks of unloading, whereas no change in muscle force was found during this period (Figure 1f-h). Furthermore, a series of in vitro studies demonstrated that myocyte-derived exosomal miR-206 could inhibit osteoblastic activity during microgravity simulation. (1) After incubating osteoblasts with the exosomes from the supernatant of MGS-C2C12 cells, the expression levels of mature miR-206 but not pri-miR-206 or pre- miR-206 in osteoblasts was elevated and the osteoblastic activity was depressed (Figure 2a-d). (2) The myocyte-derived CD63-mCherry-labeled exosomes were observed in osteoblasts after incubation with the supernatant from MGS-C2C12 cells transfected with CD63-mCherry (Figure 1e-f). (3) Up-regulated miR-206 level, but no changes in pri-miR-206 and pre-miR-206 levels within endogenous miR-206- deleted osteoblasts were observed after incubation with the supernatant from MGS-C2C12 cells (Figure 2g-h). Finally, the miR-206 knockout (KO) mice were successfully transfected with the lentiviral vector of mmu-miR-206-eGFP into the soleus (Figure 2i) and subsequently subjected to 28 day hindlimb suspension. The e-GFP-miR-206 appeared in osteoblasts from miR-206 KO mice. In addition, the elevated miR-206 level, but no changes in pri-miR-206 and pre-miR206 levels within osteoblasts associated with the depressed BFR and MAR in miR-206 KO mice (Figure 2j-k). Taken together, the applicants raised the hypothesis that skeletal muscle-derived exosomal miR-206 could inhibit osteoblastic bone formation during unloading-induced muscle atrophy.

To test the above hypothesis, the applicants have the following three specific aims: (1) To examine the effect of mmu-miR-206-eGFP transfection in soleus on miR-206 level in osteoblasts, bone formation, bone mass and bone structure in miR-206 KO mice following 28 day hind limb suspension; (2) To examine the effect of the (Asp-Ser-Ser)6 -liposome-encapsulated-antagomir-206 pre-treatment on the miR- 206 level in osteoblasts, bone formation, bone mass and bone structure from miR-206 KO mice transfected with mmu-miR-206-eGFP in soleus following 28 day hind limb suspension; (3) To examine the effect of the (Asp-Ser-Ser)6-liposome-encapsulated-antagomir-206 pre-treatment on miR-206 level in osteoblasts, bone formation, bone mass and bone structure from SD rats following 28 day hindlimb suspension.

This proposal would provide a new insight into miRNA-mediated molecular communication from skeletal muscle to bone. It would also help to generate a better understanding of how these two tissues integrate and crosstalk in both health and disease to stimulate new therapeutic strategies to enhance and maintain musculoskeletal health.