Session Type: Abstract Submissions (ACR)
Background/Purpose: A common feature of aging-related diseases, such as osteoarthritis (OA), is the progressive accumulation of damaged macromolecules leading to cell dysfunction and death. Autophagy, a key pathway for cellular homeostasis by removing such damaged molecules and organelles, including mitochondria, has a protective and survival-promoting function. Recent studies indicated that autophagy decreases with aging and OA contributing to the accumulation of damaged macromolecules. In addition, there is increasing evidence that mitochondrial dysfunction plays a critical role in accelerating the aging process and several lines of evidence demonstrated mitochondrial dysfunction in OA cartilage. The objective of this study is to determine whether activation of autophagy protects from mitochondrial dysfunction in human chondrocytes.
Methods: Human chondrocytes were treated with Rotenone (10 μg/ml), Antimycin (40 μg/ml) and Oligomycin (10 μg/ml), a mitochondrial respiratory chain (MRC) inhibitors of complex I, III and IV, respectively. Mitochondrial function and cell death were evaluated by Flow Cytometry (FC) and Fluorescence Microscopy (FM). Autophagy activation was analyzed by determination of LC3, a main marker of autophagy activation by Immunofluorescence (IF). To investigate whether autophagy protects from mitochondrial dysfunction, autophagy was induced by mTOR inhibition, using mTORC1 selective inhibitor Rapamycin (Rapa, 10 μM) and mTORC1 and mTORC2 inhibitor Torin 1 (50 nM). The effects on autophagy, mitochondrial function and chondrocyte viability were analyzed by IF, FC and FM.
Mitochondrial dysfunction was induced by 6 h treatment with MRC inhibitors, which significantly decreased mitochondrial membrane potential (Δψm) (ROT: 26.17 ± 5.9; AA: 18.21 ± 3.28; Oligo: 41.74 ± 7.59, expressed as % vs control; *p < 0.01). These results are consistent with increased ROS production (26.8 %, 44.6 % and 25.7 % for ROT, AA and Oligo, respectively; *p < 0.001) and cell death by apoptosis at 12 h (Control: 13.56 ± 1.83; ROT: 33.66 ± 5.55; AA: 29.05 ± 4.262; *p<0.05). Autophagy activity determined by LC3 expression significantly reduced in response to MRC inhibitors at 12 h. To evaluate whether autophagy regulates mitochondrial dysfunction, chondrocytes were pretreated with Rapa and Torin 1 for 4 h and then treated with the MRC inhibitors for 12 h. The results show an increase in LC3 expression compared to MRC inhibitors alone. Furthermore, autophagy inducers Rapa and Torin1 increased Δψm (Rapa: 125.8 ± 20.74; Rapa+ROT: 44.67 ± 10.37; Rapa+AA: 30.71 ± 5.949; Rapa ± Oligo: 108.5 ± 55.03 and Torin 1: 90.34 ± 9.17; Torin 1+ROT: 35.24 ± 2.7; Torin 1+AA: 41.98 ± 6.49; Torin 1+Oligo: 98.19 ± 16.81; *p<0.05), decreased ROS production (*p<0.05) and reduced cell death, suggesting a protective effect of autophagy activation on pharmacologically induced mitochondrial dysfunction.
Conclusion: These data identify autophagy activation as a protective mechanism from mitochondrial dysfunction in human chondrocytes. Pharmacological interventions that enhance autophagy may have chondroprotective activity in articular cartilage with defects on mitochondrial function.
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/