The benefits of exercise on health and longevity are well-established, and evidence suggests that these effects are partially driven by a spectrum of bioactive molecules released into circulation during exercise (e.g., exercise factors or ‘exerkines’). Recently, extracellular vesicles (EVs), including microvesicles (MVs) and exosomes or exosome-like vesicles (ELVs), were shown to be secreted concomitantly with exerkines. These EVs have therefore been proposed to act as cargo carriers or ‘mediators’ of intercellular communication. Given these findings, there has been a rapidly growing interest in the role of EVs in the multi-systemic, adaptive response to exercise. This review aims to summarize our current understanding of the effects of exercise on MVs and ELVs, examine their role in the exercise response and long-term adaptations, and highlight the main methodological hurdles related to blood collection, purification, and characterization of ELVs.

Abbreviations: ADAMTS5, ADAM metallopeptidase with thrombospondin type 1 motif 5; BHLHE40, basic helix-loop-helix family member E40; CTGF, connective tissue growth factor; Cyr61, cysteine-rich angiogenic inducer 61; FiO 2 , fraction of inspired oxygen; GM-CSF, granulocyte macrophage colony stimulating factor; Gulp1, GULP PTB domain containing engulfment adaptor 1; HIF-1, hypoxia-inducible factor-1; IFNγ, interferon gamma; IGF, insulin-like growth factor; Il, interleukin; IKzf2, IKAROS family zinc finger 2; MRF, myogenic regulatory factors; Myf5, myogenic factor 5; MyoD, myogenic differentiation; NF-κB, nuclear factor kappa B; Pax7, paired box 7; Pmp22, peripheral myelin protein 22; STAT3, signal transducer and activator of transcription 3; TAZ, Taffazin; TNFα, tumor necrosis factor alpha; YAP, yes associated protein. AbstractAcute environmental hypoxia may potentiate muscle hypertrophy in response to resistance training but the mechanisms are still unknown. To this end, twenty subjects performed a 1-leg knee extension session (8 sets of 8 repetitions at 80% 1 repetition maximum, 2-min rest between sets) in normoxic or normobaric hypoxic conditions (FiO2 14%). Muscle biopsies were taken 15 min and 4 hours after exercise in the vastus lateralis of the exercised and the non-exercised legs. Blood samples were taken immediately, 2h and 4h after exercise. In vivo, hypoxic exercise fostered acute inflammation mediated by the TNFα/NF-κB/IL-6/STAT3 (+333%, +194%, + 163% and +50% respectively) pathway, which has been shown to contribute to satellite cells myogenesis. Inflammation activation was followed by skeletal muscle invasion by CD68 (+63%) and CD197 (+152%) positive immune cells, both known to regulate muscle regeneration. The role of hypoxia-induced activation of inflammation in myogenesis was confirmed in vitro. Acute hypoxia promoted myogenesis through increased Myf5 (+300%), MyoD (+88%), myogenin (+1816%) and MRF4 (+489%) mRNA levels in primary myotubes and this response was blunted by siRNA targeting STAT3. In conclusion, our results suggest that hypoxia could improve muscle hypertrophic response following resistance exercise through IL-6/STAT3-dependent myogenesis and immune cells-dependent muscle regeneration. K E Y W O R D Sexercise, hypoxia, inflammation, muscle, myogenesis 1886 | BRITTO eT al.

Purpose: To investigate exosome-like vesicle (ELV) plasma concentrations and markers of multivesicular body (MVB) biogenesis in skeletal muscle in response to acute exercise. Methods: Seventeen healthy (BMI: 23.5±0.5kg·m-2) and fifteen prediabetic (BMI: 27.3±1.2kg·m-2) men were randomly assigned to two groups performing an acute cycling bout in normoxia or hypoxia (FiO2 14.0%). Venous blood samples were taken before (T0), during (T30) and after (T60) exercise and biopsies from m. vastus lateralis were collected before and after exercise. Plasma ELVs were isolated by size exclusion chromatography, counted by nanoparticle tracking analysis (NTA), and characterized according to international standards, followed by expression analyses of canonical ELV markers in skeletal muscle. Results: In the healthy normoxic group, the total number of particles in the plasma increased during exercise from T0 to T30 (+313%) followed by a decrease from T30 to T60 (-53%). In the same group, an increase in TSG101, CD81 and HSP60 protein expression was measured after exercise in plasma ELVs; however, in the prediabetic group, the total number of particles in the plasma was not affected by exercise. The mRNA content of TSG101, ALIX and CD9 were upregulated in skeletal muscle after exercise in normoxia; whereas, CD9 and CD81 were downregulated in hypoxia. Conclusions: ELV plasma abundance increased in response to acute aerobic exercise in healthy subjects in normoxia, but not in prediabetic subjects, nor in hypoxia. Skeletal muscle analyses suggested that this tissue did not likely play a major role of the exercise-induced increase in circulating ELVs.

Acute hypoxia has previously been suggested to potentiate resistance traininginduced hypertrophy by activating satellite cell-dependent myogenesis rather than an improvement in protein balance in human. Here, we tested this hypothesis after a 4-week hypoxic vs normoxic resistance training protocol. For that purpose, 19 physically active male subjects were recruited to perform 6 sets of 10 repetitions of a one-leg knee extension exercise at 80% 1-RM 3 times/week for 4 weeks in normoxia (FiO 2 : 0.21; n = 9) or in hypoxia (FiO 2 : 0.135, n = 10). Blood and skeletal muscle samples were taken before and after the training period. Muscle fractional protein synthetic rate was measured over the whole period by deuterium incorporation into the protein pool and muscle thickness by ultrasound. At the end of the training protocol, the strength gain was higher in the hypoxic vs the normoxic group despite no changes in muscle thickness and in the fractional protein synthetic rate. Only early myogenesis, as assessed by higher MyoD and Myf5 mRNA levels, appeared to be enhanced by hypoxia compared to normoxia. No effects were found on myosin heavy chain expression, markers of oxidative metabolism and lactate transport in the skeletal muscle. Though the present study failed to unravel clearly the mechanisms by which hypoxic resistance training is particularly potent to increase muscle strength, 2 of 15 | van DOORSLaER DE tEn RYEn Et aL.

Electrical pulse stimulation (EPS) has been suggested to be a useful method to investigate the mechanisms underlying the adaptations of human skeletal muscle to both endurance and resistance exercise. Although different myotube stimulation protocols mimicking acute and chronic endurance exercise have been developed, no convincing protocol mimicking resistance exercise exists. Adaptations to resistance exercise mainly ensue via the Akt/mTOR pathway. Therefore, the aim of this study was to develop a high frequency EPS protocol mimicking resistance exercise both acutely (100 Hz, 15 V, 0.4 ms with 4 s rest between each contraction for 30 min) and chronically (acute EPS protocol repeated on three consecutive days) on human myotubes. Compared to control conditions, the acute EPS protocol increased the phosphorylation of Akt Ser473 at 0 h (+91%, p = 0.02) and 3 h (+95%, p = 0.01), and mTOR Ser2448 at 0 h (+93%, p = 0.03), 1 h (+129%, p = 0.01), and 3 h (+104%, p = 0.0250) post-stimulation. The phosphorylation of ERK1/2 Thr202/Tyr204 was increased at 0 h (+69%, p = 0.02) and 3 h (+117%, p = 0.003) post-stimulation compared to control conditions. In addition, both S6K1 Thr389 (+157%, p = 0.009) and S6 Ser240/244 (+153%, p = 0.003) phosphorylation increased 1 h after EPS compared to control conditions. Chronic EPS protocol increased the phosphorylation of S6K1 Thr389 1 h (+105%, p = 0.03) and 3 h (+126%, p = 0.02) and the phosphorylation of S6 Ser240/244 1 h (+32%, p = 0.02) after the end of the last stimulation. In conclusion, the present work shows that human muscle cells subjected to EPS can be used as an in vitro model of acute and chronic resistance exercise.