DYNAMIC REMODELING OF MITOCHONDRIAL CALCIUM EXCHANGE DURING OSTEOBLAST DIFFERENTIATION

Bone mineralization requires coordinated calcium handling during osteoblast differentiation, yet the contribution of mitochondrial calcium transport to this process remains poorly understood. Here, we investigated how mitochondrial Ca²⁺ exchange is remodeled during osteogenesis. MC3T3-E1 cells and primary neonatal rat calvarial osteoblasts were differentiated under osteogenic conditions and analyzed for mitochondrial calcium transport and intracellular calcium homeostasis. Osteoblasts exhibited increased expression of proteins related to both mitochondrial calcium uptake (MCU) and efflux (NCLX, Tmem65), and altered ratios of regulatory proteins MICU1/MICU2, indicating remodeling of the mitochondrial Ca²⁺ transport machinery during differentiation. To investigate the functional impact of these changes, we measured mitochondrial Ca²⁺ uptake and efflux in digitonin-permeabilized cells using Calcium Green-5N fluorimetry in suspended cells. Differentiated cells exhibited increased calcium retention capacity and uptake rate compared to undifferentiated ones. Na⁺-dependent efflux rates was also increased along differentiation, consistent with the increased NCLX/Tmem65 expression. We then assessed cytosolic Ca²⁺ dynamics in Fura-2 loaded cells. Differentiated cells displayed prolonged cytosolic Ca²⁺ transients upon calcium release from the endoplasmic reticulum via thapsigargin addition, and these transients became sensitive to inhibition of mitochondrial Ca²⁺ uptake or efflux, demonstrating increased mitochondrial participation in intracellular calcium signaling. Pharmacological modulation of mitochondrial exchange in osteoblasts along the differentiation using ruthenium red (MCU inhibitor), CGP-37157 (mitochondrial Na⁺/Ca²⁺ efflux inhibitor), and morin, a MICU1-dependent activator of mitochondrial Ca²⁺ uptake was the used to investigate the influence of mitochondrial calcium exchange in osteoblast differentiation. Chronic inhibition of either mitochondrial Ca²⁺ uptake or efflux delayed extracellular matrix mineralization, whereas enhancement of mitochondrial Ca²⁺ uptake accelerated mineral deposition. Together, these findings demonstrate that mitochondrial calcium transport undergoes extensive remodeling during osteoblast differentiation and suggest that dynamic mitochondrial Ca²⁺ cycling coordinates intracellular calcium signaling required for timely extracellular matrix mineralization. These results identify mitochondrial calcium exchange as a potential regulatory hub linking calcium signaling to osteogenesis.