Robust post-translational oxidation of the mitochondrial proteome after volumetric muscle loss injury coincides with lost redox balance

The purpose of this study was to investigate a potential mechanism of mitochondrial dysfunction after volumetric muscle loss (VML) injury by assessing oxidative stress and protein post-translational oxidation (PTO). We hypothesized VML injury would result in greater redox imbalance and mitochondrial protein PTO at 7-days post-injury (dpi). Male C57BL/6J mice underwent VML injury to the hindlimb plantarflexors (gastrocnemius, soleus, plantaris muscles), and at 7-dpi mitochondrial reactive oxygen species (ROS) were assessed along with complementary total proteomic and PTO analysis of the remaining skeletal muscle. Statistical comparisons were made to uninjured muscles from age-matched naïve male mice. ROS emissions from VML-injured permeabilized myofibers were greater for both fat- (1.4-fold, p < 0.001) and carbohydrate-substrates (3.4-fold, p< 0.0001) compared to naive. Furthermore, the remaining muscle’s antioxidant buffering capacity, the ability to neutralize ROS, was significantly less in VML compared to naïve under both substrates (carb -44% and fat -29%, p < 0.01). Proteomic analysis was performed on enriched mitochondrial fractions at 7-dpi. There were 214 differentially expressed proteins at 7-dpi compared to naïve (|log2 fold-change| > 1 and FDR < 0.05). Of these differentially expressed proteins, only 14 were down-regulated while the remaining 197 were up-regulated; however, a mitochondrial pathway enrichment analysis revealed that there were 29 significant pathways (|NES| > 1.5 and FDR < 0.05), all of which were down-regulated compared to naïve. This robust down-regulation of mitochondrial protein pathways is consistent with prior transcriptomic studies. Finally, a PTO analysis was performed on the mitochondrial enriched fractions. This form of proteomics distinctly examines both reversible and irreversible PTO events (e.g. sulfenic acid and sulfonic acid). At the peptide level, there were distinct differences in the number of oxidized peptides detected between VML-injured and naïve muscles particularly evident in reversible oxidation events. The largest differences were seen in mono-oxidation events of cysteine residues (46% greater, VML = 5814 vs. naïve = 3979), followed by the reversible oxidation of methionine (40% greater, VML = 2166 vs. naïve = 1548). Irreversible tri-oxidation of cysteine was 20% greater in VML (VML = 145 vs. naïve = 121). Finally, irreversible di-oxidation of cysteine was 13% greater in VML (VML = 35 vs. naïve = 31). Of the 17,925 peptides examined for PTO after VML injury, 20% (3,511) were found to be significant (|Log2 Fold-Change| > 1 and coefficient of variance < 30%) after normalizing by protein abundance compared to naïve. Of the significant peptides, 88% (3,056) were up-regulated and 30% (1,056) are associated with the mitochondria (MitoCarta 3.0). In terms of types of oxidation events, the majority (61%) of significant peptides did not contain any oxidation event. While 28% (984) contained at least one mono-oxidation of cysteine, followed by methionine oxidation with 13% (447), and lastly tri-oxidation with 1% (35). Funding: NIH R01AR078903 (SMG and JAC). This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.