Effect of Extracellular Vehicles, Including Exercise Mimetics, for Improving Bone Loss Caused by Osteoblast Endoplasmic Reticulum Stress

Abstract

PURPOSE: This study aims to investigate the bone-muscle crosstalk mechanism by treating dysfunctional osteoblasts with extracellular vehicles (EVs) containing exercise mimetics released during muscle contraction, with the intention of identifying key factors in regulatory mechanisms.METHODS: To understand the bone-muscle crosstalk mechanism, the effects of treatment were evaluated in osteoblasts and C2C12. First, palmitate was administered to osteoblasts to induce bone loss due to endoplasmic reticulum (ER) stress. For validation, two groups were evaluated: a control group (CON) and the palmitate treatment group (PAL). Then, C2C12 myotubes were stimulated with electrical pulses to induce muscle contraction, and the released extracellular vehicles (EVs) were harvested and applied to osteoblasts. The results were used to determine the relevant mechanisms for improving bone loss. The impact of EVs released from muscles on osteoblasts was examined using the CD63-GFP plasmid. Four groups were used to compare across the different variables of interest: CON; EV, EV-treated group; PAL; EP, EV+palmitate-treated group.RESULTS: The palmitate-treated osteoblasts showed increased expression of inositol-requiring protein 1α (IRE1α), decreased sarcoplasmic/ endoplasmic reticulum Ca<sup>2+</sup>-ATPase (SERCA), reduced ER Ca<sup>2+</sup>, and mitochondrial membrane potential (MMP), indicating induced ER stress. Palmitate-induced ER stress was associated with elevated nuclear factor kappa B (NFκB) and receptor activator of NFκB (RANKL) levels and an increased RANKL/osteoprotegerin (OPG) ratio, causing bone loss. The treatment of osteoblasts with EVs from muscle contraction was found to improve ER stress, the levels of ER Ca<sup>2+</sup>, MMP, NFκB, and RANKL, and the RANKL/OPG ratio.CONCLUSIONS: Palmitate reduces ER Ca<sup>2+</sup> and induces osteoblast ER stress while impairing mitochondrial function. Furthermore, through the RANKL/RANK/OPG pathway, a key mechanism related to bone loss is induced. However, substances induced by muscle contraction can act on osteoblasts through exosomes, potentially improving this process.