THU0017 In Vitro Studies Using Cybrids Show that Mtdna Haplogroup J and H have Different Mitochondrial Activity. A Possible Explanation to OA Pathogenesis
Authors: M. Fernández-Moreno, T. Hermida-Gόmez, A. Soto-Hermida, J. Fernández-Tajes, M.E. Vázquez-Mosquera, E. Cortés-Pereira, S. Relaño-Fernández, N. Oreiro-Villar, C. Fernández-Lόpez, E. Gallardo-Pérez, R. Garesse, I. Rego-Pérez, F.J. Blanco
Journal: Annals of the Rheumatic Diseases (2015)

<h3>Background</h3> Previous studies have showed the mtDNA haplogroup J are associated with incidence and progression of OA. Transmitochondrial cybrids are optimal cellular models to study mitochondrial biology and function since they carry different mitochondrial variants with the same nuclear background, excluding those variations from the nuclear genome <h3>Objectives</h3> 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 <h3>Methods</h3> Cybrids were developed using 143B.TK<sup>-</sup> 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 lactic acid production and glucose consumption. OXPHOS function was evaluated by O<sub>2</sub> consumption (Oroboros®). The mitochondrial ROS production and percentage of apoptotic cells were measured by Flow Cytometry using DHR 123 and Annexin-V respectively. The expression levels of OA-related genes (Mn-SOD and IL-6), were evaluated by qRT-PCR. Appropriate statistical analyses were performed with GraphPad Prism v5 and qBase software <h3>Results</h3> J cybrids had higher lactic acid production than H (51.42 mg/ml and 64.22 mg/ml p&lt;0.05). Glucose consumption in No-OA cybrids revealed similar levels but higher in J than in H cybrids. O<sub>2</sub> consumption reflected that mitochondrial respiratory function in H cybrids was statistic significantly (p≤0.05) lower in all respiratory states (C<sub>r</sub>, C<sub>ro</sub> and C<sub>ru</sub>) than in J. Flux ratios did not show differences between cybrids, respiratory control ratio (RCR=C<sub>ru</sub>/C<sub>ro</sub>), uncoupling control ratio (UCR=C<sub>ru</sub>/C<sub>r</sub>) and the percentage of respiratory capacity for ATP production (%=(C<sub>r</sub>-C<sub>ro</sub>)/C<sub>ru</sub>) was similar in both cybrids. Mitochondrial ROS production was lower in J than in H (41.26±7.48; 52.21±11.38, p&lt;0.05). Apoptosis showed 2-fold increase with Staurosporine (0.2 μM, 2 hours) in H cybrids (7.35±1.7) in comparison to J (4.69±1.6) (p&lt;0.05). Baseline expression of MnSOD was higher in cybrids J than in H the same result was obtained for IL-6.Experiments performed in cybrids obtained from OA platelets showed the same behavior and confirm the results explained before <h3>Conclusions</h3> Cybrids described in this word have different metabolic behavior, being J cybrids more efficient using glucose via glycolysis. J cybrids need more O<sub>2</sub> consumption, produced less mitochondrial reactive oxygen species and are less susceptible to undergo apoptosis than H cybrids and. All these results showed that the mitochondria obtained from healthy and OA donors had a different behaviour and also these differences were increased by their own mitochondrial haplogroup. These data also offer a real rationale for why haplogroup J is associated with lower risk of OA <h3>Disclosure of Interest</h3> None declared