Hypoxic Training-induced Autophagy Is Benefit For The Improvement Of Aerobic Exercise Performance And Underlying Mechanisms

Objective:Hypoxic training is an effective strategy for improving exercise capacity.Regular exercise training under low-oxygen environment conditions can stimulate a series of physiological responses to adapt to hypoxic training,thus enhancing muscle adaptation to exercise,and improving exercise performance.However,its underlying mechanisms are still unclear.In the current study,the hypoxic cabin with oxygen concentration of 14.2%(similar with3000 m environment above sea level)for mouse model was established,so as to explore the improvement of aerobic exercise performance and underlying mechanisms or its corresponding signal pathways associated with autophagy induced by hypoxia training.Methods:Totally 90 male KM mice with the age of 8 weeks and body weight of 39.56±3.36g were randomly divided into six groups including normoxia control(NC),hypoxia control(HC),normoxia exercise(NE),hypoxic exercise(HE),hypoxia combined with chloroquine(CQ)administration at the dose of 40 mg/kgd(HQ),hypoxic exercise combined CQ administration at the dose of 40 mg/kgd(HEQ)groups with 15 mice in each group.After adapting to the new environment for 1 week,60 mice form hypoxia groups were housed at a controlled hypoxic cabin(14.2%O₂),the other mice from NC and NE groups were housed at a normoxic environment.The mice from CQ groups were administered with CQ by intraperitoneal injection once a day for 8consecutive weeks.Animal aerobic exercise training protocol:The mice from NE,HE and HQE groups were provided with 8-week swimming training with five training schedules a week and a training duration of 60 minutes under the normoxia condition.Exhaustive exercise(EE)protocol and assessment of exercise capacity(EC):The mice from EE subgroups(n=30,5 mice per group)performed a loaded swimming session.Then,the mice were subjected to swimming until exhaustion.The exhaustive swimming time was recorded for evaluating EC.After hypoxic training,BLA,HB,SOD and MDA in serum were determined for evaluating aerobic exercise capacity,in combination with the exhaustive swimming time.Histologichemical analysis and immunofluorescence of myocardial and gastrocnemius were conducted for evaluating autophagy and apoptosis.The mitochondria and the number of autophagosomes were examined by transmission electron microscope.The expression of proteins in myocardial and gastrocnemius associated with autophagy,apoptosis,oxidative stress,mitochondrial quality control and energy metabolism was evaluated by Western blot,such as Nrf2,Keap1,Parkin,Mnf2,Drp1,AMPK,phosphor-AMPK,PGC-1?,Irisin,Beclin1,LC3-I,LC3-II,p62,Bcl-2,Bax and GAPDH.Results:Hypoxic training improves aerobic performance:compared with NC group,mice from HC,NE and HE groups exhibited the significant prolongation of exhaustive swimming time.Similarly,compared with NC group,the mice from HC,NE and HE groups revealed the enhanced Hb level,increased SOD activity,and significant reduction of MDA level in serum,with the most significant effect in HE group(p<0.05).On the contrary,mice from HQ groups exhibited the decreased Hb content,reduced SOD activity,and significant increased of MDA level(p<0.05).In addition,compared with the NC group,after exhaustive swimming,BLA in serum from the HE group revealed the most rapid decrease,while BLA in serum from HQ group exhibited the most slow decrease.Through the observation by transmission electron microscope(TEM),the mitochondria in myocardial and gastrocnemius of the mice from the NC group were evenly distributed and revealed the circle or oval shape with clear mitochondrial membrane and cristae and without any fractures or typical autophagosome structures.In contrast,the mitochondria in the myocardium and gastrocnemius of mice from HC group exhibited the slightly sparsed distribution with ordered arrangement and the scattered autophagosomes.The increased expression of Drp1 and Parkin in myocardium and gastrocnemius in the mice of HC group was observed.The mitochondria in the myocardium and gastrocnemius of mice from NE group exhibited the slightly disorganized with clear mitochondrial membrane,cristae and the scattered autophagosomes.Furthemore,the structure of mitochondria in the myocardium and gastrocnemius of mice from HE group was neatly arranged.A large number of mitochondria could be observed with clear structure,mitochondrial cristae and autophagosomes.Western blot analysis showed that the expression of Mfn2,Drp1 and Parkin in the myocardium and gastrocnemius of the mice from NE and HE groups was significantly increased,but the increase of Mfn2 was the largest and the increase of Drp1 and Parkin was the smallest in the HE group.On the contrary,the disordered structure of mitochondria,broken myocardial fibers,changed size and morphology of mitochondria with swelling and cristae fracture,decreased matrix density,obvious mitochondrial vacuolation and autophagosomes in the myocardium and gastrocnemius of the mice from HQ group were observed.The expression of Mfn2 was significantly decreased,while the expression of Drp1 and Parkin in the myocardium and gastrocnemius of the mice from HQ group was significantly increased,indicating that hypoxic training promoted mitochondrial fusion in the myocardium and gastrocnemius of the mice from HE group,reduced mitochondrial division,resulted in mitophagy and significantly improved mitochondrial quality,which were greater than those in the NE group.Western blot showed that the expression of Atg7,Beclin1,LC3-II/LC3-I in myocardium and gastrocnemius of the mice form HC,NE,HE,HQ and HQE groups revealed the significant increase when compared with the NC group,while the expression of p62/SQSTM1 in myocardium and gastrocnemius of the mice from HE group exhibited the significant reduction,but increase in HQ and HQE groups.Moreover,immunofluorescence revealed the consistent results with Western blot.Therefore,both normoxic and hypoxic training could induce the increase of autophagic flux(increased LC3-II/LC3-I ratio and reduced p62)in myocardium and gastrocnemius,which was confirmed by chloroquine-blocked autophagy flux in the mice from HQ and HQE groups.The expression of Bcl-2 in myocardium and gastrocnemius of the mice from NE and HE groups increased significantly when compared with NC group,which revealed the largest increase in HE group,in contrast,the significantly down-regulated Bcl-2 and up-regulated Bax in myocardium and gastrocnemius of the mice from HQ group.The TUNEL results were basically consistent with those of Western blot,suggesting that hypoxic training is benefit for the suppression of apoptosis in the myocardium and gastrocnemius of the mice.The expression of p-AMPK/AMPK,PGC-1?and Irisin in myocardium and gastrocnemius increased in the mice form NE and HE groups,especially in the HE group when compared with that in the NC group.In contrast,the expression of p-AMPK/AMPK,PGC-1?and Irisin in myocardium and gastrocnemius significantly down-regulated in the mice form HQ group,indicating that both normoxic training and hypoxic training significantly up-regulated the phosphorylation of AMPK,thereby elevating the expression of PGC-1?and Irisin of myocardial and gastrocnemius in mice.In addition,the effect of hypoxic training was better than that of normoxic training.The expression of Nrf2 in myocardium and gastrocnemius from each group was significantly higher than that of NC group,with the largest increase in HE group.The expression of Keap1 in myocardium and gastrocnemius decreased significantly in mice from HC,NE and HE groups,but the expression of Keap1 in myocardium and gastrocnemius increased significantly in mice from HQ and HQE groups.Both normoxic training and hypoxic training could activate the expression of Nrf2 in myocardium and gastrocnemius of the mice,and the effect of hypoxic training was better than that of normoxic training.Conclusion:Hypoxic exposure at the oxygen concentration of 14.2%combined with unloaded swimming can improve aerobic exercise capacity,which may be due to the increased energy metabolism,enhanced antioxidant stress capacity,improved mitochondrial quality and suppressed apoptosis and hypoxic training-induced autophagy activation through activating AMPK/PGC-1?/Nrf2 signaling pathway.