Altered oxidative DNA damage repair systems in muscles of old mice: role in the age-related increase in muscle production of cytokines/chemokines.

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An increased presence of the oxidised DNA base lesion 8-oxo-7,8-dihydroguanine (8-oxoG) is seen in muscles of old mice. 8-oxoG is repaired by the 8-oxoguanine DNA glycosylase-1 (OGG1)-initiated DNA base excision repair pathway, a process proposed to lead to activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB). We hypothesised that chronic oxidative DNA damage in muscles of old mice leads to the chronic increase in activation of NF-kB by OGG1 and the subsequent production of inflammatory cytokines by skeletal muscle, contributing to age-related deterioration of muscle function.An in vitro model of oxidative DNA damage was established by treating C2C12 myoblasts with H2O2. DNA damage repair responses including 8-oxoG, OGG1, PARP-1, NF-kB activation and cytokine/chemokine gene expression and release by muscle cells were determined. The effect of TH5487 (5uM), a selective OGG1 inhibitor on NF-kB activation and cytokine production was determined. Isolated muscle fibres from adult mice were treated in a similar manner to myoblasts described above and examined for the release of cytokines/chemokines. The role of OGG1 in the chronic production of cytokines by isolated muscle fibres of old mice was examined using Luminex multiplex analysis. The effect of TH5487 on H2O2-mediated cytokine release by fibres of adult mice and on chronic release of cytokines/chemokines by fibers of old mice was examined.Cell viability was maintained in myoblasts treated with 50uM H2O2 but Ogg1 mRNA levels were significantly increased as was acetylation of OGG1, PARP-1 and cleaved PARP-1 protein levels. Activation of NF-kB was evident and accompanied by an increased expression and release of a number of pro-inflammatory cytokines (3.7 fold, 4.2 fold, 3 fold increases in IL-6 TNF-a and CXCL1 mRNA respectively). These H2O2-mediated increase in NF-kB activation and increase in cytokine/chemokine mRNA levels were normalized when cells were pretreated with TH5487 prior to treatment with H2O2.Experiments using isolated muscle fibers from adult mice demonstrated an increase in NF-kB activation following treatment with H2O2. This H2O2-mediated increase in NF-kB DNA binding activity was significantly decreased in muscle fibers pretreated with TH5487. Isolated muscle fibers from old compared with adult mice showed an increase in cytokine/chemokine release which was also reduced by treatment with TH5487. Increases in IL-6, CCL2, CXCL1, CCL7, GM-CSF, CX3CL1,CCL27,CXCL5,CCL11, CXCL16 and CXCL-12 were seen in the media of fibres from adult mice following treatment with H2O2 and IL-6,CCL2, CXCL1,CCL7, CCL27, CXCL5,CCL11,CXCL16 and CXCL-12 were decreased to normal values by pretreatment with TH5487. Isolated muscle fibers from old mice showed a significant increase in IL-6, CXCL1, CCL2, CCL7, of which all were reduced following treatment with TH5487, suggesting that OGG1 pathway may be responsible, at least in part for the increased production of cytokines/chemokines by muscle of old mice. Data on proteomic and mRNA analyses of muscles from adult and old mice for evidence of changes in DNA repair pathways with age will also be presented. A computational model of DNA damage repair is being designed to identify DNA damage and repair process therapeutic targets to modify activation of NF-kB in muscles of old mice.MRC-Arthritis Research UK Centre for Integrated Research into Musculoskeletal Ageing (CIMA)