Drp1-Mediated Mitochondrial Fission Coordinates Mineral Precursor Transport and Mitochondrial Homeostasis During Osteogenesis

BACKGROUND During the osteogenic differentiation of human mesenchymal stem cells (hMSCs), amorphous calcium phosphate (ACP) precursors within mitochondria are transported to the extracellular matrix via PINK1/Parkin-mediated mitochondrial autophagy, thereby initiating mineralization. However, the mechanisms by which mitochondria maintain functional homeostasis in the face of such extensive consumption remain to be elucidated. The present study investigated the role of Drp1-mediated mitochondrial fission in coordinating ACP transport and maintaining mitochondrial function. MATERIAL AND METHODS hMSCs were treated with shRNA to knock down Drp1 or with pharmacological inhibitors (Mdivi-1, CsA). Mitochondrial morphology, ACP transport, energy metabolism, and osteogenic differentiation were assessed using transmission electron microscopy, ATP assays, qPCR, western blot, and eosin staining. In vivo skeletal mineralization phenotypes were validated using a zebrafish model. RESULTS Following osteogenic induction, Drp1 Ser616 phosphorylation increased while Ser637 phosphorylation decreased; mitochondrial fission increased, producing fragments approximately 450 nm in diameter, matching the size of autolysosomes, which facilitates ACP encapsulation and clearance. Inhibition of Drp1 resulted in abnormal mitochondrial elongation, ACP accumulation, and reduced ATP production (decreased to 59.2% in the sh-Drp1 group), and significantly suppressed RUNX2 and ALPL expression as well as mineralization (mineralization area decreased to 36.7%). In zebrafish, treatment with Mdivi-1 or CsA similarly led to reduced craniofacial mineralization and downregulation of osteogenic gene expression. CONCLUSIONS Drp1-mediated mitochondrial fission strikes a balance between the efficient transport of ACP precursors and mitochondrial functional homeostasis via the fission-clearance-regeneration cycle. This process serves as a key regulatory node in osteogenic differentiation. Further investigation of Drp1-associated mitochondrial dynamics in bone metabolic disease models is warranted.