Skeletal muscle conveys several of the health-promoting effects of exercise; yet the underlying mechanisms are not fully elucidated. Studying skeletal muscle is challenging due to its different fiber types and the presence of non-muscle cells. This can be circumvented by isolation of single muscle fibers. Here, we develop a workflow enabling proteomics analysis of pools of isolated muscle fibers from freeze-dried human muscle biopsies. We identify more than 4000 proteins in slow- and fast-twitch muscle fibers. Exercise training alters expression of 237 and 172 proteins in slow- and fast-twitch muscle fibers, respectively. Interestingly, expression levels of secreted proteins and proteins involved in transcription, mitochondrial metabolism, Ca²⁺ signaling, and fat and glucose metabolism adapts to training in a fiber type-specific manner. Our data provide a resource to elucidate molecular mechanisms underlying muscle function and health, and our workflow allows fiber type-specific proteomic analyses of snap-frozen non-embedded human muscle biopsies.

Key points: Low‐volume high‐intensity exercise training promotes muscle mitochondrial adaptations that resemble those associated with high‐volume moderate‐intensity exercise training. These training‐induced mitochondrial adaptations stem from the cumulative effects of transient transcriptional responses to each acute exercise bout. However, whether metabolic stress is a key mediator of the acute molecular responses to high‐intensity exercise is still incompletely understood. Here we show that, by comparing different work‐matched low‐volume high‐intensity exercise protocols, more marked metabolic perturbations were associated with enhanced mitochondrial biogenesis‐related muscle mRNA responses. Furthermore, when compared with high‐volume moderate‐intensity exercise, only the low‐volume high‐intensity exercise eliciting severe metabolic stress compensated for reduced exercise volume in the induction of mitochondrial biogenic mRNA responses. The present results, besides improving our understanding of the mechanisms mediating exercise‐induced mitochondrial biogenesis, may have implications for applied and clinical research that adopts exercise as a means to increase muscle mitochondrial content and function in healthy or diseased individuals. The aim of the present study was to examine the impact of exercise‐induced metabolic stress on regulation of the molecular responses promoting skeletal muscle mitochondrial biogenesis. Twelve endurance‐trained men performed three cycling exercise protocols characterized by different metabolic profiles in a randomized, counter‐balanced order. Specifically, two work‐matched low‐volume supramaximal‐intensity intermittent regimes, consisting of repeated‐sprint (RS) and speed endurance (SE) exercise, were employed and compared with a high‐volume continuous moderate‐intensity exercise (CM) protocol. Vastus lateralis muscle samples were obtained before, immediately after, and 3 h after exercise. SE produced the most marked metabolic perturbations as evidenced by the greatest changes in muscle lactate and pH, concomitantly with higher post‐exercise plasma adrenaline levels in comparison with RS and CM. Exercise‐induced phosphorylation of CaMKII and p38 MAPK was greater in SE than in RS and CM. The exercise‐induced PGC‐1α mRNA response was higher in SE and CM than in RS, with no difference between SE and CM. Muscle NRF‐2, TFAM, MFN2, DRP1 and SOD2 mRNA content was elevated to the same extent by SE and CM, while RS had no effect on these mRNAs. The exercise‐induced HSP72 mRNA response was larger in SE than in RS and CM. Thus, the present results suggest that, for a given exercise volume, the initial events associated with mitochondrial biogenesis are modulated by metabolic stress. In addition, high‐intensity exercise seems to compensate for reduced exercise volume in the induction of mitochondrial biogenic molecular responses only when the intense exercise elicits marked metabolic perturbations.

Purpose: To examine the degree of neuromuscular fatigue development along with changes in muscle metabolism during two work-matched high-intensity intermittent exercise protocols in trained individuals.Methods: In a randomized, counter-balanced, crossover design, 11 endurance-trained men performed high-intensity intermittent cycle exercise protocols matched for total work and including either multiple short-duration (18 × 5 s; SS) or long-duration (6 × 20 s; LS) sprints. Neuromuscular fatigue was determined by preexercise to postexercise changes in maximal voluntary contraction force, voluntary activation level and contractile properties of the quadriceps muscle. Metabolites and pH were measured in vastus lateralis muscle biopsies taken before and after the first and last sprint of each exercise protocol.Results: Peak power output (11% ± 2% vs 16% ± 8%,P< 0.01), maximal voluntary contraction (10% ± 5% vs 25% ± 6%,P< 0.05), and peak twitch force (34% ± 5% vs 67% ± 5%,P< 0.01) declined to a lesser extent in SS than LS, whereas voluntary activation level decreased similarly in SS and LS (10% ± 2% vs 11% ± 4%). Muscle [phosphocreatine] before the last sprint was 1.5-fold lower in SS than LS (P< 0.001). Preexercise to postexercise intramuscular accumulation of lactate and H⁺was twofold and threefold lower, respectively, in SS than LS (P< 0.001), whereas muscle glycogen depletion was similar in SS and LS. Rate of muscle glycolysis was similar in SS and LS during the first sprint, but twofold higher in SS than LS during the last sprint (P< 0.05).Conclusions: These findings indicate that, in endurance-trained individuals, multiple long-sprints induce larger impairments in performance along with greater degrees of peripheral fatigue compared to work-matched multiple short-sprints, with these differences being possibly attributed to more extensive intramuscular accumulation of lactate/H⁺and to lower rates of glycolysis during multiple long-sprint exercise.