Fast activation/deactivation of the NFκB signalling pathway in human skeletal muscle: role of oxygenation and metabolite accumulation

Abstract

INTRODUCTION: The NFκB signalling pathway plays a critical role in inflammation, immunity, cell proliferation, apoptosis and muscle metabolism and is activated by extracellular signals and intracellular changes in Ca2+, Pi, H+, metabolites and reactive oxygen and nitrogen species (RONS). Studies in rodents have reported NFκB activation by exercise, with the scarce data in humans reporting contradicting findings. Cell culture experiments have shown that NFκB and STAT3 are stimulated by hypoxia, which exacerbates RONS production. However, it remains unknown whether metabolite accumulation, muscle oxygenation and hypoxia influence NFκB signalling in response to acute exercise in human skeletal muscle. We hypothesized that RONS production during incremental exercise to exhaustion (IE) would upregulate NFκB signalling depending on metabolite accumulation, with a more exacerbated response in Hypoxia (Hyp) than normoxia (Nx). METHODS: Eleven active men performed IE to exhaustion in Nx and Hyp (PIO2:73 mmHg). Immediately after IE, the circulation of one leg was instantaneously occluded (300 mmHg). Muscle biopsies from m. vastus lateralis were taken before (PRE), and 10s (POST, occluded leg) and 60s after exercise from the occluded (OC1M) and non-occluded (nOC1M) legs simultaneously, and blood samples were taken throughout from the femoral vein. Protein expression of key markers in the NFκB and MAPK signalling pathways (Western Blot) and muscle metabolites (fluorometry) were measured. Statistical analysis was performed with repeated-measures ANOVA. RESULTS: At post, muscle lactate augmented 25% solely in OC1M (P<0.05) while PCr was reduced by 94 and 48% in OC1M and nOC1M, respectively (P<0.005) regardless of PIO2. PO2 in the femoral vein was 21.1±2.0 and 10.6±2.8 mmHg at Wmax, in Nx and Hyp, respectively (P<0.001). The ratios pTyr705/Total STAT3 and pSer176-180/Total IKKβ, pSer536 NFκB p65, and the total amount of NFκB p65, p50 and p105 NFκB were significantly elevated at POST, collectively indicating activation of NFκB. This was facilitated by the phosphorylation of IkBb at Thr19- Ser23, which releases its inhibitory action on NFκB. Post-exercise ischemia maintained these changes (OC1M), while these signals were reverted to the pre-exercise condition after one minute of recovery with free circulation. The expression of IL-6 and the phosphorylation state of ERK1/2 and p38 did not change significantly. All responses were similar regardless of exercise PIO2. CONCLUSION: This study shows that NFκB signalling is activated in human skeletal muscle to a similar degree during incremental exercise to exhaustion in normoxia and severe hypoxia. The fact that post-exercise ischemia maintained the activation of NFκB suggests that reoxygenation after exercise is necessary to deactivate NFκB. Our results indicate that the metabolites accumulated during the exercise or the lack of O2 may play a role in maintaining NFκB signalling.