Fig. 4: Metabolite alterations in muscle of PolG^mut mice mediated by muscle specific loss of PolG exonuclease activity.

Unbiased assessment of metabolite abundance was performed in gastrocnemius muscle of PolG^cont and PolG^mut mice with (A) pathway enrichment analysis (MetaboAnalyst-KEGG) undertaken in short-term and (B) long-term cohorts, where metabolic pathways are listed in order of significance, red gradient indicating P-value, and the size of the circle denoting the enrichment ratio. C Metabolites in PolG^mut short term (red) and long term (blue) skeletal muscle that had greater than 0.05-fold change (relative to PolG^cont). D Integrated metabolic pathway analysis (MetaboAnalyst) of short-term skeletal muscle bulk RNA-sequencing and metabolomics data sets, with red gradient indicating P-value and circle size indicating pathway impact. Metabolomics data (A–D) was normalised by median and log transformed using MetaboAnalyst 5.0, (short-term: n = 8/group & long-term: PolG^cont n = 8, PolG^mut n = 7). E relevant folate cycle genes detected by RNA-sequencing (n = 6/group) and (F) proteins detected by mitochondrial proteomics, n = 5/group. Analyses of transcriptomic data (E) and proteomic data (F) were corrected using Benjamini-Hochberg FDR. G Schematic of the mammalian cytosolic/mitochondrial folate cycle (red text denotes upregulated proteins and blue indicating reduced folate intermediates) in short-term cohort. Targeted metabolomics was performed to determine folate cycle metabolic intermediates in (H) short (PolG^cont n = 10, PolG^mut n = 9) and (I) long-term (PolG^cont n = 8, PolG^mut n = 7) cohorts (reduced 5,10-Methenyl-THF highlighted in short term folate cycle schematic in blue). E, F, H, I data are presented as mean ± SEM, with P-value between control and mutant biological replicates determined by two-sided, unpaired two-tailed t-test (E, F) or Mann-Whitney test (H, I). THF = Tetrahydrofolate; TMP = Thymidine monophosphate; ζm5U = 5-taurinomethyluridine. Source data for these figures are provided in the Source Data file. Fig. 4G Created in BioRender. Drew, B. (2025) https://BioRender.com/t82k138.