Role Of AMPK In Changes Of Mitochondrial Turnover And Microcirculation In Skeletal Muscle Upon Hypoxic Training

Part ?: Hypoxic training upregulates AMPK activity,mitochondrial turnover,and microcirculation in skeletal musclePurpose: AMPK positively regulates the mitochondrial turnover(biogenesis and mitophagy).Improvement of muscle microcirculation function by hypoxic training may be related to mitochondrial turnover mediated by AMPK.Thus,this part investigated the effects of hypoxic exposure,exercise training,and hypoxic training on the AMPKmediated mitochondrial turnover and microcirculation.Methods: Male C57BL/6 mice were randomly assigned to control(C),hypoxic exposure(H),exercise training(E),live high training low(LHTL),and live low training high(LLTH)groups,n = 8/ per group.5 days/week for early 3 weeks and 6 days/week for later 3 weeks during six-week interventions.C group did not perform any intervention;H group was exposed to hypoxia 8 h everyday(14.8%-12.5% oxygen concentration from the first to the last week);E group underwent incremental load training(10 m/min × 30 min of first week to 20 m/min × 80 min of last week,0° incline);LHTL completed the combined loads from H and E groups;LLTH exercised everyday with the E group's load and additional hypoxic stimulus simultaneously.Laser doppler flowmeter,western blot,and immunohistochemistry were used to evaluate blood perfusion,protein of gastrocnemius,and angiogenesis of quadriceps.Results: Microcirculation and angiogenesis: Cutaneous microcirculatory blood perfusion(MBP)and heating cutaneous microcirculatory blood perfusion(H-MBP)of H group were higher than pre-values(p < 0.05).Immunohistochemistry VEGF level of H group was higher than C group(p < 0.05).MBP,H-MBP,and muscle microcirculatory blood perfusion(M-MBP)of each group from E,LHTL,and LLTH were higher than pre-values(p < 0.05).Immunohistochemistry CD31,VEGF levels of LHTL and LLTH groups were higher than C group(p < 0.05).Mitochondrial turnover and energy metabolism of gastrocnemius: Compared with C group,AMPK?(Thr172)phosphorylation expression did not change in H group(p > 0.05).H group's PGC-1?and Pink1/Parkin proteins also did not alter(p > 0.05).ATP content,citrate synthase(CS),and ATP synthase(ATPase)activities of H group increased significantly(p < 0.05).E group's AMPK?(Thr172)phosphorylation,AMPK? expressions were enhanced significantly(p < 0.05),but p38 MAPK phosphorylation,PGC-1?,Tfam,and mitophagy signaling did not change(p > 0.05).ATP content,CS,and cytochrome c oxidase(COX)activities of E group increased significantly(p < 0.05).Compared with C group,AMPK?(Thr172),AMPK?,p38 MAPK phosphorylation,and PGC-1? levels of LHTL and LLTH groups increased significantly(p < 0.05).COX ?(LHTL)and Cyt c(LLTH)also increased significantly(p < 0.05).LHTL and LLTH also upregulated Pink1/Parkin,Nix mitophagy signaling,and ATP production(ATP content,CS,COX,and ATPase activities)markedly(p < 0.05).Conclusion: Hypoxic training activated AMPK and mitochondrial turnover signaling in skeletal muscle,paralleled with the increases of angiogenesis and microcirculation blood perfusion,which was superior than hypoxic exposure or normoxic trainingPart ?: Mitochondrial turnover and microcirculation adaptations in skeletal muscle by AMPK activation were similar to those by hypoxic trainingPurpose: This part was to compare the features including mitochondrial turnover and microcirculation function between AMPK activation and hypoxic training intervention,determining whether there were similarities in the adaptations brough by these two interventions.Methods: Male C57BL/6 mice were randomly assigned to control(C),hypoxic training(H),AMPK activation(E),and AMPK activation + hypoxic training(AH)groups,n = 8/per group.5 days/week for early 3 weeks and 6 days/week for later 3 weeks during six-week interventions.A group underwent intraperitoneal injection of AICAR(250 mg/kg).H group completed LHTL intervention(identical with Part ?).AH group performed combined intervention from A and H group.Other methods were identical with Part ?.Results: Microcirculation and angiogenesis: MBP,H-MBP and M-MBP of each group from A,H,and AH groups were higher than pre-values except of A group's M-MBP(p< 0.05).Immunohistochemistry CD31,VEGF levels of H and AH groups,and VEGF level of A group were higher than C group(p < 0.05).Mitochondrial turnover and energy metabolism of gastrocnemius: Compared with C group,AMPK?(Thr172),AMPK?,mitochondrial biogenesis(p38 MAPK phosphorylation and PGC-1?)in H,A and AH group increased significantly(p < 0.05).COX ?(H),Cyt c(AH),and mitophagy proteins(Pink,Parkin,and Nix)of H and AH groups also increased significantly(p <0.05).CS and ATPase activities of A group,ATP content,CS,and ATPase activities of H and AH groups increased significantly(p < 0.05).Conclusion: AMPK activation intervention upregulated mitochondrial turnover and microcirculation of skeletal muscle,which was similar to the results after hypoxic training intervention.Part ?: AMPK?2 deletion inhibits the increased responses of mitochondrial turnover and microcirculation in skeletal muscle by hypoxic trainingPurpose: After AMPK?2 muscle specific deletion,we studied whether the inhibition of AMPK was paralleled with repressions of mitochondrial turnover and the microcirculation function.Methods: Male muscle AMPK?2 specific knockout mice and wild type mice were randomly assigned to wild control(WC),wild hypoxic training(WH),Knockout control(KC),and Knockout hypoxic training(KH)groups,n = 7/ per group.5 days/week for early 3 weeks and 6 days/week for later 4 weeks during the seven-week interventions.WH and KH performed the same LHTL protocol(10 m/min × 30 min + 13.7% oxygen concentration exposure of first week to 24 m/min × 80 min + 12.5% oxygen concentration exposure of last week).WC and KC groups kept sedentary condition.Other methods were identical with Part ?.Results: Microcirculation and angiogenesis: Compared with WC group,MBP,H-MBP,and M-MBP of WH group were increased(p < 0.01),while there was no obvious difference between KC and KH groups in these variables(p > 0.05).Immunohistochemistry CD31 expression of WH group was higher than that of WC group(p < 0.01),but this result did not exist between KC and KH groups(p > 0.05).Mitochondrial turnover and energy metabolism of gastrocnemius: Compared with WC group,AMPK?(Thr172),AMPK?,p38 MAPK phosphorylation increased significantly(p < 0.01).Sirt1,whole cell Tfam,and Cyt c did not exhibit any difference among these groups(p > 0.05).PGC-1?,mitochondrial Tfam,and COX ? increased dramatically in WH group compared with WC group(p < 0.01),while there was no any difference between KC and KH groups in these proteins(p > 0.05).Hypoxic training enhanced Pink1 expression independent of genotype(WC vs.WH,KC vs.KH,p < 0.01).Whole cell Parkin,Nix,and mitochondrial Parkin were higher in WH group than WC group(p< 0.05),but these results above mentioned can not be observed between KC and KH groups.The same results also existed in term of ATP content,CS,COX,and ATPase activities.Conclusion: Muscle specific deletion of AMPK?2 gene inhibited the increased adaptions of mitochondrial turnover and microcirculation in skeletal muscle after hypoxic training.AMPK is involved with the increases of mitochondrial turnover and microcirculation of skeletal muscle upon hypoxic training.