Application of post-exercise ischemia reveals an important role of pi and po2 in the regulation of nrf2 and nfkb signalling in human skeletal muscle

Abstract

Exercise-induced signaling is mediated by changes in intracellular Ca2+, Pi, H+, metabolites, PO2, cell energy, and ROS. Nevertheless, it remains unknown which of these plays a predominant role. Ischemia application immediately after maximal exercise traps the accumulation of Pi, metabolites and H+, maintaining PO2 low. One min after exercise with free circulation recovery, Pi and PO2 are largely recovered. We hypothesized signaling depending on increased Pi and reduced PO2 will be maintained and that depending on Ca2+ transients would be reduced during ischemia. To test this, we measured, in muscle biopsies, Nrf2 and NFkB signaling (ROS dependent), and CaMKII (ROS and Ca2+ dependent), before and after incremental exercise to exhaustion (IE) and post-exercise ischemia. METHODS: Eleven physically active men performed IE in Normoxia (Nx) and Hypoxia (Hyp) (PIO2:73 mmHg). Immediately after IE, the circulation of one leg was instantaneously occluded (300 mmHg). Protein expression (Western Blot) and muscle metabolites were measured in m. vastus lateralis biopsies before (PRE), and 10 (POST, solely occluded leg) and 60s after exercise from the occluded (OC1M) and non-occluded (nOC1M) legs. Blood was sampled from the femoral vein. Statistics: repeated-measures ANOVA. RESULTS: At POST, muscle lactate increased only at OC1M (25%; P<0.05) and PCr was reduced by 94 and 48% in OC1M and nOC1M, respectively (P<0.005), similarly in Nx and Hyp. Femoral vein PO2 was 21.1±2.0 and 10.6±2.8 mmHg at Wmax, in Nx and Hyp, respectively (P<0.001). At POST, pThr286-CaMKII, pSer40-Nrf2, p105-NFkB, and p50-NFkB were elevated, and Keap1 reduced. One min after exercise, Thr286-CaMKII, pSer40-Nrf2, p105 and p50-NFkB and Keap1 remained at POST level in the occluded leg, while they recovered to PRE values in the nonoccluded leg. pSer32/36-IkBa was reduced by 29% at POST remaining reduced in OCM1 (P<0.01), while it recovered to PRE values in OC1M. Total IkBa was reduced 1min after exercise by 45%, regardless of occlusion. Despite increased protein carbonylation in Hyp, signaling responses were similar in Nx and Hyp (P>0.05). CONCLUSION: This study shows that pThr286-CaMKII, NFkB and Nrf2 signaling pathways are activated during IE to a similar extent in Nx and Hyp. Although femoral vein PO2 was much lower during IE in Hyp and the oxidative stress higher, the signaling responses were similar in Nx and Hyp. Interestingly, just one min after exercise observed phosphorylations returned to basal, but only when recovery occurred with reperfusion. Furthermore, Keap1 was rapidly resynthesized with free circulation, suggesting that post-exercise biopsies should be taken immediately to capture fast-responding kinases and allosteric regulators. The fact that the occlusion maintained Thr286-CaMKII, Nrf2 and NFkB signaling active suggests that low PO2 or increased Pi play a critical role in activating and maintaining these signals.