Subcellular proteomic profiling of human skeletal muscle reveals exercise-induced coordinated and compartment-specific protein remodeling

Abstract Exercise training induces extensive protein modifications in skeletal muscle, yet how acute exercise and training-induced molecular responses are spatially coordinated across muscle subcellular compartments remains unclear. Using subcellular fractionation combined with data-independent acquisition mass spectrometry, we profiled skeletal muscle mitochondrial, nuclear, and cytosolic proteomes in response to an acute bout of intense cycling (pre-, mid-, post- and 3 h post-exercise) and after eight weeks of endurance training in 40 healthy adults (20 males and 20 females). Acute exercise triggered coordinated, compartment-specific proteomic remodelling, including reductions in protein translation and import machinery concomitant with increased redox-related proteins. Notably, acute exercise increased markers of ribosomal translation within the mitochondrial fraction, revealing ribosomal scaffold protein RACK1 as a potential regulator of subcellular translational control under contractile stress (confirmed by targeted immunoblotting). The nuclear proteome displayed transient remodelling of RNA-processing and chromatin-associated proteins, while cytosolic changes were modest. Endurance training induced robust proteomic remodelling across all compartments, including increased markers of mitochondrial oxidative metabolism and proteostasis. While there were sex differences at baseline, subcellular proteomic responses were largely conserved between sexes. We provide the first comprehensive, time-course subcellular characterisation of the skeletal muscle proteome, revealing regulation of translational machinery underlying the acute exercise response. Competing Interest Statement The authors have declared no competing interest.