Mitophagic activity and protein levels differ across and within muscles: implications for future skeletal muscle mitophagy research

Skeletal muscle is a heterogeneous tissue consisting of fibers with distinct contractile speeds, metabolic profiles, and cellular signaling. This heterogeneity may extend to mitochondrial quality control processes such as mitophagy. Using mt-Keima mice, we found that mitophagic activity was greater in the fast-twitch, glycolytic extensor digitorum longus (EDL) compared to the slow-twitch, oxidative soleus (SOL) muscle. Live imaging of quadriceps (QUAD) muscle revealed two distinct fiber populations: those with high total mt-Keima signal but low mitophagic activity, and others with low signal but higher mitophagic activity. Additionally, we observed skeletal muscle type and regional differences in autophagic and mitophagic protein content. Further, select mitophagic proteins strongly correlated with mitochondrial proteins across different regions of the gastrocnemius, while others did not. These findings highlight the complexity of mitophagy regulation in skeletal muscle and emphasize the importance of considering muscle phenotype, including fiber type, region, and mitochondrial content when studying mitophagy.Abbreviations: AIFM1: apoptosis inducing factor mitochondria associated 1; ATG: autophagy related; ATG7: autophagy related 7; BNIP3: BCL2 interacting protein 3; BNIP3L: BCL2 interacting protein 3 like; BCL2L13: BCL2 like 13; CSA: cross-sectional area; CYCS: cytochrome c, somatic; EDL: extensor digitorum longus; FUNDC1: FUN14 domain containing 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GAS: gastrocnemius; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MYH: myosin heavy chain; OXPHOS: oxidative phosphorylation; PINK1: PTEN induced kinase 1; PLANT: plantaris; PRKN: parkin RBR E3 ubiquitin protein ligase; QUAD: quadriceps; SLC25A4: solute carrier family 25 member 4; SOD2: superoxide dismutase 2; SOL: soleus; SQSTM1: sequestosome 1; TFAM: transcription factor A, mitochondrial; VDAC1: voltage dependent anion channel 1.