Session Type: Abstract Submissions (ACR)
Autophagy, is a key pathway of cellular homeostasis for removing damaged macromolecules and organelles, including mitochondria. Recent studies indicate that autophagy activation is defective in aging and osteoarthritis (OA), contributing to the cell death and tissue damage. In addition, there is increasing evidence that mitochondrial dysfunction plays an important role in OA pathogenesis. The objective of this study is to determine whether activation of autophagy protects from mitochondrial dysfunction in human chondrocytes.
Human chondrocytes were treated with Oligomycin (10 μg/ml), a mitochondrial respiratory chain (MRC) inhibitor of complex V. Mitochondrial function and cell death were evaluated by Flow Cytometry, Fluorescence Microscopy. Autophagy activation was analyzed by determination of LC3-II, a main marker of autophagy activation by Immunofluorescence and Western Blot. To investigate whether autophagy protects from mitochondrial dysfunction, autophagy was induced by mammalian target of rapamycin complex 1 (mTORC1) selective inhibitor Rapamycin (Rapa, 10 μM) and the dual mTORC1 and mTORC2 inhibitor Torin 1 (50 nM). Genetic deletion of Atg5 (siAtg5), a essential autophagy marker for autophagosome formation, was employed to evaluate the role of autophagy in mitochondrial dysfunction.
Mitochondrial dysfunction was induced by treatment with Oligomycin, which significantly decreased mitochondrial membrane potential (Δψm) (Oligo: 41.74 ± 7.59, expressed as % vs control; *p < 0.01). This was associated with increased intracellular ROS production (25.7 % vs. control; *p < 0.001 compared to control condition) and mitochondrial superoxide generation (29.61 % vs. control; *p < 0.001 compared to control condition). Furthermore, increased cell death by apoptosis was observed (Control: 11.35 ± 1.735; Oligo: 25.37 ± 6.767, *p<0.05 vs. control). Autophagy activation determined by LC3-II was increased at short incubation times, perhaps acting as an early response to stress and then decrease in a time dependent manner. To evaluate whether autophagy regulates mitochondrial dysfunction, chondrocytes were pretreated with Rapa and Torin 1 and then treated with Oligomycin. The results show an increase in LC3 expression compared to MRC inhibitor alone. Furthermore, autophagy inducers Rapa and Torin1 increased Δψm (Rapa: 125.8 ± 20.74; Rapa ± Oligo: 108.5 ± 55.03 and Torin 1: 90.34 ± 9.17; Torin 1+Oligo: 98.19 ± 16.81; *p<0.05), decreased ROS production and reduced cell death (*p<0.05), suggesting a protective effect of autophagy activation on pharmacologically induced mitochondrial dysfunction. Importanly, blocking autophagy by siAtg5 showed a significant dysfunctional changes in the Δψm and ROS production (*p<0.05), indicating the essential role of autophagy in mitocondrial function.
Our data highlight the role of autophagy as a critical protective mechanism against mitochondrial dysfunction. Pharmacological interventions that enhance autophagy may have chondroprotective activity in cartilage degenerative processes such as OA.
P. López de Figueroa,
F. J. Blanco,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/autophagy-activation-protects-from-mitochondrial-dysfunction-in-human-chondrocytes-2/