Understanding the endocrine crosstalk between bone and muscle: molecular investigation of the impact of myokines on osteogenesis using C2C12 myoblast and 2T3 osteoblast cell lines

Bones and skeletal muscles interact mechanically to allow locomotion in vertebrate and even invertebrate organisms. Until the last decade of research, the interactions between them had been gathered under the umbrella of the “mechanical coupling” theory, where muscles are the load suppliers and bones provide the attachment sites [1]. However, bone and skeletal muscle have recently been identified as endocrine organs, that secrete cytokines and chemokines, through which they interact to promote locomotion. This molecular and biochemical interplay has been named “bone-muscle crosstalk”. The bi-directional flow of signals between bone and muscle has been investigated experimentally by differentiating bone or skeletal muscle progenitor cells in a medium conditioned by myotubes or osteocytes respectively [2][3]. These studies have demonstrated that osteocyte (osteokines) and myotube (myokines) secreted factors have an inhibitory influence on myogenesis and osteogenesis respectively, since they reduce the majority of the mRNA levels of genes associated with differentiation. We propose to study the effects of myokines on osteogenesis by differentiating 2T3 osteoblastic cells in a medium conditioned by either early (3-5 days) or late (7-10 days) myo-tubes. We will then analyze mRNA and protein levels of marker genes of differentia-tion, to establish the effect of early and late patterns of myokines. Besides, we will characterize the differentiation process from a functional point of view by studying alkaline phosphatase activity and the deposition of mineralized matrix. As expected results, early and late myotube-conditioned media should affect differently the osteoblast lineage in the course of differentiation. If this is the case, we will proceed with a metabolomic profiling of the conditioned medium, to identify the cytokines most abundantly expressed. This first set of results will pave the way for further experiments of myoblast and osteoblast co-culture aimed at a real-time tracking of the bi-directional signaling betweeen these tissues and its impact on all stages of differentiation. The results of this study will deepen our understanding of how the muscle secretome protects osteocytes and preserve their function and vice versa how bone factors maintain muscle function. Such knowledge will help identify potential new therapies for bone and muscle diseases, especially when they co-exist, as is the case of the twin syndrome of osteoporosis and sarcopenia.