Elizabeth G. Reisman

Reactive oxygen species are essential signalling molecules that regulate numerous aspects of skeletal muscle physiology. These effects are mediated through redox post‐translational modifications on protein cysteine thiols, which influence the structure and function of redox‐sensitive proteins. Mass spectrometry‐based redox proteomic approaches have greatly advanced our ability to detect and characterise cysteine redox modifications, revealing a broad network of redox‐sensitive proteins and pathways in skeletal muscle. Recent methodological developments enable quantification of the stoichiometry of reversible oxidative modifications at specific cysteine residues, providing critical insight into the extent and functional relevance of site‐specific redox regulation. Redox proteomic approaches are being employed to improve our understanding of the specific redox protein modifications underlying physiological and pathophysiological processes in skeletal muscle. This review summarises current proteomic strategies for quantifying redox post‐translational modifications and their application to studying redox signalling in skeletal muscle. Emerging experimental approaches that offer the potential to study the specific roles of site‐specific redox modifications in muscle physiology are also discussed. Collectively, these technologies present exciting opportunities to define the mechanistic roles of individual cysteine residues in muscle biology and help uncover new therapeutic avenues for conditions characterised by impaired redox homeostasis.

Postmenopausal women represent the fastest-growing demographic at risk of sarcopenia and cardiometabolic disease, yet exercise biology research remains disproportionately derived from male or hormone-replete phenotypes. Menopause constitutes a chronic endocrine perturbation characterized by sustained reductions in estrogen and progesterone, and altered androgen balance, superimposed on the acute and chronic perturbations induced by exercise. This hormonal shift modifies substrate metabolism, inflammation, redox balance, and recovery capacity, factors that shape molecular responses to exercise across tissues and time. Here, we synthesize current evidence on exercise responses in postmenopausal females across genomics, epigenomics, transcriptomics, proteomics, and metabolomics/lipidomics. Across omics layers, direct data in postmenopausal cohorts remain limited, with frequent underreporting of menopausal status, hormone therapy exposure, circulating hormone concentrations, medication use, and biosampling timing relative to exercise and hormone dosing. We outline a menopause-aware framework for exercise-omics that prioritizes endocrine stratification, repeated sampling across exercise and recovery, and integrative multi-omics approaches linking molecular responses to functional outcomes. We also outline minimum reporting standards to improve reproducibility, inclusivity, and translational relevance. Advancing menopause-aware exercise-omics will be essential for developing precision exercise strategies that improve health span and functional independence in later life.

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.

Elucidating the time-dependent transcriptional response of skeletal muscle to exercise is essential for uncovering the molecular mechanisms that drive its health-promoting effects. However, previous studies have been limited by a small number of muscle biopsies, often collected at arbitrary time points post-exercise, and in predominantly male subjects. Using the most comprehensive skeletal muscle biopsy time course following a single session of exercise in both males and females, we identified over 16,600 differentially expressed genes (DEGs), including more than 7,000 novel exercise-responsive genes. Of these DEGs, the magnitude of differential expression in 60% of genes was influenced by cardiorespiratory fitness. Although most mitochondrial genes were differentially expressed after exercise, the majority were downregulated at 24–48 hours, suggesting that mitochondrial protein expression may be regulated by post-transcriptional regulation. Despite 1,193 genes showing sex-specific expression at baseline, exercise-induced gene expression differences were minimal between males and females, suggesting that when cardiorespiratory fitness and exercise stimulus are matched, skeletal muscle adaptations are similar between sexes. To enhance data accessibility, we created an interactive Shiny app ( https://BishopLab.shinyapps.io/EXERgene/ ) that allows users to investigate specific genes and interrogate potential mechanisms of skeletal muscle adaptation to exercise.

Analyses of mitochondrial adaptations in human skeletal muscle have mostly used whole-muscle samples, where results may be confounded by the presence of a mixture of type I and II muscle fibres. Using our adapted mass spectrometry-based proteomics workflow, we provide insights into fibre-specific mitochondrial differences in the human skeletal muscle of men before and after training. Our findings challenge previous conclusions regarding the extent of fibre-type-specific remodelling of the mitochondrial proteome and suggest that most baseline differences in mitochondrial protein abundances between fibre types reported by us, and others, might be due to differences in total mitochondrial content or a consequence of adaptations to habitual physical activity (or inactivity). Most training-induced changes in different mitochondrial functional groups, in both fibre types, were no longer significant in our study when normalised to changes in markers of mitochondrial content.

Mitochondria are essential, membrane-enclosed organelles that consist of similar to 1100 different proteins, which allow for many diverse functions critical to maintaining metabolism. Highly metabolic tissues, such as skeletal muscle, have a high mitochondrial content that increases with exercise training. The classic western blot technique has revealed training-induced increases in the relatively small number of individual mitochondrial proteins studied (similar to 5% of the >1100 proteins in MitoCarta), with some of these changes dependent on the training stimulus. Proteomic approaches have identified hundreds of additional mitochondrial proteins that respond to exercise training. There is, however, surprisingly little crossover in the mitochondrial proteins identified in the published human training studies. This suggests that to better understand the link between training-induced changes in mitochondrial proteins and metabolism, future studies need to move beyond maximizing protein detection to adopting methods that will increase the reliability of the changes in protein abundance observed.

Exercise perturbs energy homeostasis in skeletal muscle and engages integrated cellular signalling networks to help meet the contraction-induced increases in skeletal muscle energy and oxygen demand. Investigating exercise-associated perturbations in skeletal muscle signalling networks has uncovered novel mechanisms by which exercise stimulates skeletal muscle mitochondrial biogenesis and promotes whole-body health and fitness. While acute exercise regulates a complex network of protein post-translational modifications (e.g. phosphorylation) in skeletal muscle, previous investigations of exercise signalling in human and rodent skeletal muscle have primarily focused on a select group of exercise-regulated protein kinases [i.e. 5ʹ adenosine monophosphate-activated protein kinase (AMPK), protein kinase A (PKA), Ca2⁺/calmodulin-dependent protein kinase (CaMK) and mitogen-activated protein kinase (MAPK)] and only a small subset of their respective protein substrates. Recently, global mass spectrometry-based phosphoproteomic approaches have helped unravel the extensive complexity and interconnection of exercise signalling pathways and kinases beyond this select group and phosphorylation and/or translocation of exercise-regulated mitochondrial and nuclear protein substrates. This review provides an overview of recent advances in our understanding of the molecular events associated with acute endurance exercise-regulated signalling pathways and kinases in skeletal muscle with a focus on phosphorylation. We critically appraise recent evidence highlighting the involvement of mitochondrial and nuclear protein phosphorylation and/or translocation in skeletal muscle adaptive responses to an acute bout of endurance exercise that ultimately stimulate mitochondrial biogenesis and contribute to exercise’s wider health and fitness benefits.

Analyses of mitochondrial adaptations in human skeletal muscle have mostly used whole-muscle samples, where results may be confounded by the presence of a mixture of type I and II muscle fibres. Using our adapted mass spectrometry-based proteomics workflow, we provide new insights into fibre-specific mitochondrial differences in human skeletal muscle before and after training. Our findings challenge previous conclusions regarding the extent of fibre-type-specific remodelling of the mitochondrial proteome and highlight that most baseline differences in mitochondrial protein abundances between fibre types reported by us, and others, might be due to differences in total mitochondrial content or a consequence of adaptations to habitual physical activity (or inactivity). Most training-induced changes in different mitochondrial functional groups, in both fibre types, were stoichiometrically linked to changes in markers of mitochondrial content.