The exogenous antioxidants vitamin C (ascorbate) and vitamin E (α-tocopherol) often blunt favorable cell signaling responses to exercise, suggesting that redox signaling contributes to exercise adaptations. Current theories posit that this antioxidant paradigm interferes with redox signaling by attenuating exercise-induced reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation. The well-documented in vitro antioxidant actions of ascorbate and α-tocopherol and characterization of the type and source of the ROS/RNS produced during exercise theoretically enable identification of redox-dependent mechanisms responsible for the blunting of favorable cell signaling responses to exercise. This review aimed to apply this reasoning to determine how the aforementioned antioxidants might attenuate exercise-induced ROS/RNS production. The principal outcomes of this analysis are (1) neither antioxidant is likely to attenuate nitric oxide signaling either directly (reaction with nitric oxide) or indirectly (reaction with derivatives, e.g., peroxynitrite); (2) neither antioxidant reacts appreciably with hydrogen peroxide, a key effector of redox signaling; (3) ascorbate but not α-tocopherol has the capacity to attenuate exercise-induced superoxide generation; and (4) alternate mechanisms, namely pro-oxidant side reactions and/or reduction of bioactive oxidized macromolecule adducts, are unlikely to interfere with exercise-induced redox signaling. Out of all the possibilities considered, ascorbate-mediated suppression of superoxide generation with attendant implications for hydrogen peroxide signaling is arguably the most cogent explanation for blunting of favorable cell signaling responses to exercise. However, this mechanism is dependent on ascorbate accumulating at sites rich in NADPH oxidases, principal contributors to contraction-mediated superoxide generation, and outcompeting nitric oxide and superoxide dismutase isoforms. The major conclusions of this review are: (1) direct evidence for interference of ascorbate and α-tocopherol with exercise-induced ROS/RNS production is lacking; (2) theoretical analysis reveals that both antioxidants are unlikely to have a major impact on exercise-induced redox signaling; and (3) it is worth considering alternate redox-independent mechanisms.

Exercise redox biochemistry is of considerable interest owing to its translational value in health and disease. However, unaddressed conceptual, methodological and technical issues complicate attempts to unravel how exercise alters redox homeostasis in health and disease. Conceptual issues relate to misunderstandings that arise when the chemical heterogeneity of redox biology is disregarded: which often complicates attempts to use redox-active compounds and assess redox signalling. Further, that oxidised macromolecule adduct levels reflect formation and repair is seldom considered. Methodological and technical issues relate to the use of out-dated assays and/or inappropriate sample preparation techniques that confound biochemical redox analysis. After considering each of the aforementioned issues, we outline how each issue can be resolved and provide a unifying set of recommendations. We specifically recommend that investigators: consider chemical heterogeneity, use redox-active compounds judiciously, abandon flawed assays, carefully prepare samples and assay buffers, consider repair/metabolism, use multiple biomarkers to assess oxidative damage and redox signalling.

Two-dimensional gel electrophoresis provides robust comparative analysis of skeletal muscle, but this technique is laborious and limited by its inability to resolve all proteins. In contrast, orthogonal separation by SDS-PAGE and reverse-phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) affords deep mining of the muscle proteome, but differential analysis between samples is challenging due to the greater level of fractionation and the complexities of quantifying proteins based on the abundances of their tryptic peptides. Here we report simple, semi-automated and time efficient (i.e., 3 h per sample) proteome profiling of skeletal muscle by 1-dimensional RPLC electrospray ionisation tandem MS. Solei were analysed from rats (n = 5, in each group) bred as either high- or low-capacity runners (HCR and LCR, respectively) that exhibited a 6.4-fold difference (1,625 ± 112 m vs. 252 ± 43 m, p < 0.0001) in running capacity during a standardized treadmill test. Soluble muscle proteins were extracted, digested with trypsin and individual biological replicates (50 ng of tryptic peptides) subjected to LC-MS profiling. Proteins were identified by triplicate LC-MS/MS analysis of a pooled sample of each biological replicate. Differential expression profiling was performed on relative abundances (RA) of parent ions, which spanned three orders of magnitude. In total, 207 proteins were analysed, which encompassed almost all enzymes of the major metabolic pathways in skeletal muscle. The most abundant protein detected was type I myosin heavy chain (RA = 5,843 ± 897) and the least abundant protein detected was heat shock 70 kDa protein (RA = 2 ± 0.5). Sixteen proteins were significantly (p < 0.05) more abundant in HCR muscle and hierarchal clustering of the profiling data highlighted two protein subgroups, which encompassed proteins associated with either the respiratory chain or fatty acid oxidation. Heart-type fatty acid binding protein (FABPH) was 1.54-fold (p = 0.0064) more abundant in HCR than LCR soleus. This discovery was verified using selective reaction monitoring (SRM) of the y5 ion (551.21 m/z) of the doubly-charged peptide SLGVGFATR (454.19 m/z) of residues 23–31 of FABPH. SRM was conducted on technical replicates of each biological sample and exhibited a coefficient of variation of 20%. The abundance of FABPH measured by SRM was 2.84-fold greater (p = 0.0095) in HCR muscle. In addition, SRM of FABPH was performed in vastus lateralis samples of young and elderly humans with different habitual activity levels (collected during a previous study) finding FABPH abundance was 2.23-fold greater (p = 0.0396) in endurance-trained individuals regardless of differences in age. In summary, our findings in HCR/LCR rats provide protein-level confirmation for earlier transcriptome profiling work and show LC-MS is a viable means of profiling the abundance of almost all major metabolic enzymes of skeletal muscle in a highly parallel manner. Moreover, our approach is relatively more time efficient than techniques relying on orthogonal separations, and we demonstrate LC-MS profiling of the HCR/LCR selection model was able to highlight biomarkers that also exhibit differences in trained and untrained human muscle.

Acute exercise increases reactive oxygen and nitrogen species generation. This phenomenon is associated with two major outcomes: (1) redox signalling and (2) macromolecule damage. Mechanistic knowledge of how exercise-induced redox signalling and macromolecule damage are interlinked is limited. This review focuses on the interplay between exercise-induced redox signalling and DNA damage, using hydroxyl radical (·OH) and hydrogen peroxide (H2O2) as exemplars. It is postulated that the biological fate of H2O2 links the two processes and thus represents a bifurcation point between redox signalling and damage. Indeed, H2O2 can participate in two electron signalling reactions but its diffusion and chemical properties permit DNA oxidation following reaction with transition metals and ·OH generation. It is also considered that the sensing of DNA oxidation by repair proteins constitutes a non-canonical redox signalling mechanism. Further layers of interaction are provided by the redox regulation of DNA repair proteins and their capacity to modulate intracellular H2O2 levels. Overall, exercise-induced redox signalling and DNA damage may be interlinked to a greater extent than was previously thought but this requires further investigation.

High intensity training (HIT) is effective at improving health; however, it is unknown whether HIT also improves physical function. This study aimed to determine whether HIT improves metabolic health and physical function in untrained middle aged individuals. Fourteen (three male and eleven female) untrained individuals were recruited (control group n = 6: age 42 ± 8 y, weight 64 ± 10 kg, BMI 24 ± 2 kg·m⁻² or HIT group n = 8: age 43 ± 8 y, weight 80 ± 8 kg, BMI 29 ± 5 kg·m⁻²). Training was performed twice weekly, consisting of 10 × 6-second sprints with a one minute recovery between each sprint. Metabolic health (oral glucose tolerance test), aerobic capacity (incremental time to exhaustion on a cycle ergometer) and physical function (get up and go test, sit to stand test and loaded 50 m walk) were determined before and after training. Following eight weeks of HIT there was a significant improvement in aerobic capacity (8% increase in VO₂ peak; p < 0.001), physical function (11%–27% respectively; p < 0.05) and a reduction in blood glucose area under the curve (6% reduction; p < 0.05). This study demonstrates for the first time the potential of HIT as a training intervention to improve skeletal muscle function and glucose clearance as we age.

Traditional methods for phenotyping skeletal muscle (e.g., immunohistochemistry) are labor-intensive and ill-suited to multixplex analysis, i.e., assays must be performed in a series. Addressing these concerns represents a largely unmet research need but more comprehensive parallel analysis of myofibrillar proteins could advance knowledge regarding age- and activity-dependent changes in human muscle. We report a label-free, semi-automated and time efficient LC-MS proteomic workflow for phenotyping the myofibrillar proteome. Application of this workflow in old and young as well as trained and untrained human skeletal muscle yielded several novel observations that were subsequently verified by multiple reaction monitoring (MRM). We report novel data demonstrating that human ageing is associated with lesser myosin light chain 1 content and greater myosin light chain 3 content, consistent with an age-related reduction in type II muscle fibers. We also disambiguate conflicting data regarding myosin regulatory light chain, revealing that age-related changes in this protein more closely reflect physical activity status than ageing per se. This finding reinforces the need to control for physical activity levels when investigating the natural process of ageing. Taken together, our data confirm and extend knowledge regarding age- and activity-related phenotypes. In addition, the MRM transitions described here provide a methodological platform that can be fine-tuned to suite multiple research needs and thus advance myofibrillar phenotyping.