Increased muscle contractile activity, as observed with regular exercise, prevents oxidative stress—induced muscle wasting, at least partially, by improving the antioxidant defense system. Phosphorylated p62/sequestosome1 competitively binds to the Kelch‐like ECH‐associated protein 1, activating nuclear factor erythroid 2—related factor 2 (Nrf2), which stimulates transcription of antioxidant/electrophile responsive elements. However, it remains to be determined if this process is activated by regular exercise in skeletal muscle. Here, we demonstrate that muscle contractile activity increases antioxidants, Nrf2 translocation into nuclei, and Nrf2 DNA‐binding activity in association with increased p62 phosphorylation (Ser351) in mouse oxidative skeletal muscle. Skeletal muscle‐specific loss of Nrf2 [ i.e ., Nrf2 muscle‐specific knockout (mKO) mice] abolished the expression of the Nrf2 target antioxidant gene NAD(P)H‐quinone oxidoreductase 1 (NQO1) in both glycolytic and oxidative muscles but reduced exercise‐mediated increases of antioxidants ( i.e ., copper/zinc superoxide dismutase (SOD) and extracellular SOD only in oxidative muscle. Interestingly, skeletal muscle—specific loss of p62 ( i.e ., p62 mKO mice) also abolished the expression of NQO1 and reduced exercise‐mediated increases of the same antioxidants in soleus muscle. Collectively, these findings indicate that p62 and Nrf2 cooperatively regulate the exercise‐mediated increase of antioxidants in oxidative muscle.—Yamada, M., Iwata, M., Warabi, E., Oishi, H., Lira, V. A., Okutsu, M. p62/SQSTM1 and Nrf2 are essential for exercise‐mediated enhancement of antioxidant protein expression in oxidative muscle. FASEB J. 33, 8022–8032 (2019). www.fasebj.org

Chemokines are critical mediators of angiogenesis in several physiological and pathological conditions; however, a potential role for muscle-derived chemokines in exercise-stimulated angiogenesis in skeletal muscle remains poorly understood. Here, we postulated that the chemokine stromal cell-derived factor-1 (SDF-1α/C-X-C motif chemokine ligand 12: CXCL12), shown to promote neovascularization in several organs, contributes to angiogenesis in skeletal muscle. We found that CXCL12 is abundantly expressed in capillary-rich oxidative soleus and exercise-trained plantaris muscles. CXCL12 mRNA and protein were also abundantly expressed in muscle-specific peroxisome proliferator-activated receptor γ coactivator 1α transgenic mice, which have a high proportion of oxidative muscle fibers and capillaries when compared with wild-type littermates. We then generated CXCL12 muscle-specific knockout mice but observed normal baseline capillary density and normal angiogenesis in these mice when they were exercise trained. To get further insight into a potential CXCL12 role in a myofiber-endothelial cell crosstalk, we first mechanically stretched C2C12 myotubes, a model known to induce stretch-related chemokine release, and observed increased CXCL12 mRNA and protein. Human umbilical vein endothelial cells (HUVECs) exposed to conditioned medium from cyclically stretched C2C12 myotubes displayed increased proliferation, which was dependent on CXCL12-mediated signaling through the CXCR4 receptor. However, HUVEC migration and tube formation were unaltered under these conditions. Collectively, our findings indicate that increased muscle contractile activity enhances CXCL12 production and release from muscle, potentially contributing to endothelial cell proliferation. However, redundant signals from other angiogenic factors are likely sufficient to sustain normal endothelial cell migration and tube formation activity, thereby preserving baseline capillary density and exercise training-mediated angiogenesis in muscles lacking CXCL12.

CXC-chemokine receptor 4 (CXCR4) and its ligand, stromal-derived factor 1α (SDF-1α; also known as CXCL12), are crucial for the redistribution of immune cells after acute exercise. We investigated the relationships between acute exercise and CXCR4 expression on natural killer (NK) cells. Peripheral blood mononuclear cells (PBMCs) were cultured with cortisol and analyzed for CXCR4 expression on CD3⁻/CD56⁺NK cells and NK cell migration activity. To determine the effect of exercise, we isolated PBMCs from subjects before and after a 90-min exercise at 70% peak O₂uptake (V̇o_2peak) and determined the changes in CXCR4 expression on NK cells after exercise. We cultured PBMCs with plasma obtained before and after exercise and with the glucocorticoid antagonist RU-486 to determine NK cell migration activity and the effects of cortisol on CXCR4 expression in vitro. Cortisol treatment increased CXCR4 expression ( P < 0.05) and migration activity ( P < 0.05) of NK cells. Exercise did not affect CXCR4 expression on NK cells, whereas incubating them with postexercise plasma significantly increased CXCR4 expression ( P < 0.05) and migration activity ( P < 0.05). RU-486 blocked cortisol-induced CXCR4 upregulation on NK cells, but only partially blocked (7%) CXCR4 upregulation when PMBCs were incubated with postexercise plasma. Thus acute exercise increases CXCR4 expression on NK cells and their migration activity and may contribute to NK cell redistribution after acute exercise; however, cortisol did not appear to be the primary mediator of augmented CXCR4 expression.