Session Title: Genetics, Genomics and Proteomics II
Session Type: Abstract Submissions (ACR)
Mitochondria play an important role in the OA pathogenesis. mtDNA haplogroup J is significantly associated with a lower risk of OA in northwest Spanish populations. Transmitochondrial cybrids, that carry the same nuclear background and different mitochondrial variants, are optimal cellular model to study mitochondrial biology and function. This cellular model excludes variations from the nuclear genome in the cellular activity. The aim of this work is to test the real role of mtDNA haplogroups in cellular activity, using cybrids with mtDNA haplogroup H and J and analyzing several parameters implicated in the OA process
Cybrids were developed using the 143B.TK– rho-0 cell line and platelets from healthy (without OA) and OA donors with mtDNA haplogroups H and J. The metabolic status was evaluated by measuring both lactic acid production and glucose consumption. The respiration was evaluated with a high resolution respirometry (Oroboros). The ATP levels were obtained by luciferase reaction. The expression levels of genes implicated in inflammation (COX-2 and iNOS), Metalloproteinases (MMP-1, 3 and 13) and MnSOD, were evaluated by RT-PCR. The ROS production and percentage of apoptotic cells were measured by Flow Cytometry (mean fluorescence intensity).
Cybrids carrying the mtDNA haplogroup J show higher lactic acid production (62.22 mg/ml and 52.71 mg/ml; p<0.05) and 20% higher of glucose consumption than H cybrids, therefore being more efficient using glucose vie glycolysis, In addition, J cybrids show lower levels of ATP (0.027nmol ATP/mg protein) than H (0.033nmol ATP/mg protein), and higher values of oxygen consumption (36.92 nmmol/ml for J cybrids and 9.97 nmmol/ml for H cybrids). H cybrids had significantly higher levels of total ROS (203.30 for H cybrids and 131.26 for J) and mitochondrial ROS (47.36 and 36.87 respectively). MnSOD expression in basal conditions was higher in cybrids H than J (2-fold) and IL-1β stimulation (5 ng/ml 24 hours) showed 2-fold increase of MnSOD in J cybrids compared to H. The analysis of inflammatory process showed that the basal expression levels of COX-2 and iNOS were higher in H than in J (H expressed 4-fold COX-2 than J; iNOS was 1.5-fold). Basal expression of MMP-1, 3 and 13 was higher in cybrids H than J. The percentage of cell in spontaneous apoptosis was similar between cybrids H (3.76%) and J (5.78%). The use of staurosporine (0.2µM, 2 hours) to induce apoptosis showed a 7-fold increase of apoptosis in H cybrids.
Experiments performed in OA cybrids confirm the metabolic differences between H cybrids and J cybrids, as well as the higher susceptibility of H cybrids than to undergo apoptosis
H and J cybrids have different metabolic behavior (J are more glycolysis dependent than H). Cybrids J have a lower ATP production, lower inflammatory response and produce less reactive species of oxygen. Cybrids J are less susceptible to undergo apoptosis. All these results offer a real rationale for why haplogroup J is associated with lower risk of OA
M. Fernandez Moreno,
M. E. Vázquez-Mosquera,
F. J. Blanco Garcia,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/transmitocondrial-cybrids-a-tool-to-study-the-role-of-mtdna-haplogroups-in-oa-pathogenesis/