Fig. 2: Molecular and functional specific changes in mitochondria induced by muscle specific loss of PolG exonuclease activity.

Protein abundance in mitochondria from skeletal muscle of PolG^cont and PolG^mut short term mice was analysed using proteomics. Computational analysis of the data was performed including (A) hierarchical clustering, (B) principal component analysis (PCA) and, (C) volcano plot depicting proteins that were significantly altered, (low=blue dots and high=red dots) in PolG^mut mitochondria compared to PolG^cont (grey dots=not significantly altered). D Enrichment analysis of proteins significantly regulated in mitochondria between the genotypes, depicting upregulated pathways in red, and downregulated pathways in blue. Size of the circle indicates the number of proteins that contribute to the cluster. E Volcano plot of differentially regulated proteins between the genotypes, with the individual proteins of the Electron Transport Chain (ETC Complex I-V) highlighted (CI = green, CII = Red, CII = pink, CIV = purple, CV = orange) and (F) Volcano plot highlighting proteins from the mitochondrial ribosome complex (blue dots). Analyses of proteomic data (A–F) were corrected using Benjamini-Hochberg FDR, n = 8/group. Isolated mitochondria were also analysed for functional differences including (G) Blue Native PAGE gel with immunoblot for Complex I (NDUFA9) and Complex II (SDHA) formation and, (H) quantitation of BN-PAGE blots, n = 4/group. The Seahorse analyser was used on fresh mitochondria isolated from muscle to measure Oxygen Consumption Rate (OCR) for activity of Complex I (CI), Complex II (CII) and Complex IV (CIV) from (I, J) short-term cohort (PolG^cont n = 6, PolG^mut n = 9), and (K, L) long-term cohort (PolG^cont n = 6, PolG^mut n = 5). Lipidomics was performed in TAs of PolG^cont and PolG^mut from long-term cohorts demonstrating the (M) abundance of the cardiolipin pre-cursor lipid phosphatidylglycerol (PG), and (N)total cardiolipin (CL), PolG^cont n = 6, PolG^mut n = 5. I-N are presented as mean ± SEM, with P-value between control and mutant biological replicates determined by two-sided, unpaired two-tailed t-test (H) or Mann-Whitney test (I–N). O Abundance of individual CL species (fold change from PolG^cont) in TA and Liver from PolG^cont and PolG^mut long-term mice (purple = increased, green = decreased in PolG^mut), PolG^cont n = 6, PolG^mut n = 5. Source data for these figures are provided in the Source Data file.