| BackgroundLiving high-training low(HiLo)is widely used in sports training as it can improve oxygen transport capacity while effectively increasing maximal oxygen uptake and improving aerobic capacity.Hypoxic training,including HiLo,increases antioxidant capacity and improves cardiopulmonary function.This training method promotes a simultaneous increase in oxygen transport in the cardiovascular system and oxygen consumption in skeletal muscle,creating a new dynamic equilibrium.Hypoxic training promotes skeletal muscle oxygen transport by improving skeletal muscle microcirculatory function.It also promotes skeletal muscle oxygen consumption levels by facilitating improved skeletal muscle mitochondrial function.Skeletal muscle microcirculation and mitochondria maintain an appropriate oxygen concentration gradient between skeletal muscle cells and the immediately adjacent microvasculature.The combination of improved microcirculatory function and mitochondrial function in skeletal muscle results in skeletal muscle adaptation.However,the underlying mechanisms by which HiLo regulate changes in skeletal muscle microcirculatory vessels and mitochondrial quality control are unclear.Silent mating type information regulation 2 homolog 3(Sirt3)can influence skeletal muscle mitochondrial quality control by modulating downstream biological signals and improving the oxidative state of mitochondria.Additionally,Sirt3 can ebgabce endothelial diastolic capacity and vascular density.It has been shown that hypoxia and exercise can promote Sirt3 protein expression.Therefore,it is hypothesized that Sirt3 may have a role in the regulation of microcirculatory changes and mitochondrial quality control in skeletal muscle by HiLo.PurposeBy inhibiting and activating Sirt3 activity,this study aimed to investigate whether Sirt3 plays a role in regulating the changes of skeletal muscle microcirculation and mitochondrial quality control with HiLo exercise.Methods Part Ⅰ: The role of living high-training low in regulating skeletal muscle microcirculation changes and mitochondrial quality control.Male C57BL/6J mice were randomly assigned to a control(C),hypoxic(H),exercise(E),living low-training high(LoHi),or living high-training low(HiLo)group,n=6/per group.The mice in the C group did not receive any treatment.The mice in the H group lived in a hypoxic environment for 8 hours per day,with oxygen concentrations decreasing from 14.8% in the first week to 12.5% in the sixth week(corresponds to an approximate altitude of 2740 – 3960 m).The mice in the E group performed aerobic exercise on a treadmill once a day.From the first week to the sixth week,the speeds of the treadmill were 10,12,14,16,18,and 20 m/min,respectively.The duration of the training increased from 30 min to 80 min from the first to the six week,with a weekly increase of 10 min.The mice in the LoHi group lived in normoxia and exercised under hypoxic conditions once a day.The oxygen concentration during exercise was the same as in the H group and the intensity and duration of exercise was the same as in the E group.The mice in the HiLo group lived in a hypoxic environment and exercised under normoxia once a day.The hypoxic conditions were the same as in group H.The intensity and duration of exercise were the same as in the E group.After 6 weeks of intervention,mouse weight and microcirculatory perfusion levels indexes in the skeletal muscle including MBP,H-MBP,M-MBP were detected.The protein level of skeletal muscle vascular endothelial diastolic capacity and vascular density indexes(eNOS,AT1 R,AT2R,CD31),mitochondrial quality control indexes(p-p38 MAPK,p38 MAPK,PGC-1α,PINK1,Parkin,Bnip3,Mfn1,Mfn2,Opa1,Drp1),Sirt3 and its downstream indexes(FOXO3a,AC-SOD2),and mitochondrial antioxidant indexes(Prx3,Prx5,Trx2)were measured by Western Blot.Skeletal muscle oxidative and antioxidant indicators CAT,GSH-Px,SOD,and MDA were examined.The morphological characteristics of skeletal muscle mitochondria were observed by transmission electron microscopy.Part Ⅱ: Effects of Sirt3 inhibition intervention on the changes in skeletal muscle microcirculation and mitochondrial quality control with the regulation of living high-training lowMale C57BL/6J mice were randomly assigned to a C,HiLo,Sirt3 inhibition(3-TYP),or 3-TYP + HiLo group,n=6/per group.The mice in the C group did not receive any treatment.The mice in the HiLo group underwent HiLo exercise for 6 weeks.The mice were housed at an oxygen concentration of 13.8%(corresponding to an approximate altitude of 3350m)for 8 hours per day and then subjected to normoxic training.The treadmill speed for the normoxic training increased from 10 m/min to 20 m/min gradually,with a weekly increase of 2m/min per week.The duration of the training increased from 30 min to 80 min from the first to the six week,with a weekly increase of 10 min.The mice in the 3-TYP group were intraperitoneally injected with50mg/kg·2d of the 3-TYP for 6 weeks.The mice in the 3-TYP+HiLo group received HiLo after intraperitoneal injection of 3-TYP.After 6 weeks of intervention,mouse weight,microcirculatory perfusion level indexes in skeletal muscle including MBP,HMBP,M-MBP were detected.Protein levels of Sirt3 and its downstream indexes(FOXO3a,SOD2,AC-SOD2),mitochondrial antioxidant indexes(Prx3,Prx5,Trx2),skeletal muscle vascular endothelial diastolic capacity and vascular density indexes(eNOS,AT1 R,AT2R,CD31,VEGF),mitochondrial quality control indexes(p-p38 MAPK,p38 MAPK,PGC-1α,COX Ⅳ,Cytc,PINK1,Parkin,Bnip3,Mfn2,Opa1,Fis,MFF)were measured by Western Blot.Serum ET-1 levels were measured by ELISA.Skeletal muscle oxidative and antioxidant indexes(CAT,GSH-Px,SOD,MDA),mitochondrial function indexes(ATP content,CS activity),and endothelial diastolic capacity indexes(NO)were examined.The morphological characteristics of skeletal muscle mitochondria were observed using transmission electron microscopy.Part Ⅲ: Effects of Sirt3 activation intervention on the changes in skeletal muscle microcirculation and mitochondrial quality control with the regulation of living high-training low.Male C57BL/6J mice were randomly assigned to a C,HiLo,Sirt3 activation(HNK),or HNK+HiLo group,n=6/per group.The mice in the C group did not receive any treatment.The mice in HiLo group underwent HiLo for 6 weeks(same as in Part II).The mice in the HNK group were intraperitoneally injected with 10mg/kg·2d of the HNK for 6 weeks.The mice in the HNK+HiLo group received HiLo after intraperitoneal injection of HNK.After 6 weeks of intervention,mouse weight,microcirculatory perfusion level indexes in skeletal muscle including MBP,H-MBP,and M-MBP were detected.Protein level of Sirt3 and its downstream indexes(FOXO3a,SOD2,AC-SOD2),mitochondrial antioxidant indexes(Prx3,Prx5,Trx2),skeletal muscle vascular endothelial diastolic capacity and vascular density indexes(eNOS,AT1 R,AT2R,CD31,VEGF),mitochondrial quality control indexes(p-p38 MAPK,p38 MAPK,PGC-1α,COX Ⅳ,Cytc,PINK1,Parkin,Bnip3,NIX,Mfn1,Mfn2,Opa1,Fis,MFF)were measured by Western Blot.Serum ET-1 levels were measured by ELISA.Skeletal muscle oxidative and antioxidant indexes(CAT,GSH-Px,SOD,and MDA),mitochondrial function indexes(ATP content,CS activity),endothelial diastolic capacity indexes(NO)were examined.Results Part Ⅰ: The role of living high-training low in regulating skeletal muscle microcirculation changes and mitochondrial quality control.(1)In terms of skeletal muscle microcirculation,the MBP,H-MBP and M-MBP levels significantly increased following the H,E,LoHi and HiLo interventions(p < 0.05).(2)In terms of skeletal muscle vascular endothelial function and vascular density,significant increases in the eNOS and AT2 R protein expression were observed in both LoHi and HiLo groups(p < 0.01).CD31 expression were significantly increased in the H,E,LoHi and HiLo groups(p < 0.01).(3)In regards to mitochondrial quality control in skeletal muscle,the mitochondrial biogenesis factor p-p38 MAPK was significantly elevated in the E,LoHi and HiLo groups(p < 0.05,p < 0.05,p < 0.01),and the eNOS expression in the HiLo group was significantly higher than the E and LoHi groups(p < 0.05).The protein expression of PGC-1α increased only in the HiLo group(p < 0.01).Mitophagy proteins PINK1 and Parkin were significantly elevated in the LoHi and the HiLo groups(p < 0.05).The mitochondrial fusion indicators Mfn2 and Opa1 were significantly higher in the LoHi and HiLo groups(p < 0.05).(4)In terms of Sirt3 downstream signals and the levels of oxidation and antioxidant in skeletal muscle,the protein expression of Sirt3 and FOXO3 a was elevated in LoHi and HiLo groups(p < 0.05),and the acetylation level of SOD2 was decreased(p < 0.05).The CAT and GSH-Px levels significantly increased(p < 0.01,p < 0.05)and the MDA levels were significantly decreased(p < 0.05)in the HiLo group.The interventions in the E,LoHi and HiLo groups all increased the protein expression levels of Prx5 and Trx2(p < 0.01).Part Ⅱ: Effects of Sirt3 inhibition intervention on the changes in skeletal muscle microcirculation and mitochondrial quality control with the regulation of living high-training low.(1)In terms of Sirt3 downstream signals and the levels of oxidation and antioxidant in skeletal muscle,the expression of Sirt3,FOXO3 a and SOD2 in the 3-TYP+HiLo group was significantly lower than that in the HiLo group(p < 0.01).The CAT,GSH-Px,SOD and Prx5,and Trx2 expression in the 3-TYP+HiLo group were also significantly lower than that in the HiLo group(p < 0.05).(2)In regards to skeletal muscle microcirculation,the MBP,H-MBP and M-MBP level in the HiLo group were significantly higher than the C group(p < 0.05,p < 0.01,p <0.05).The H-MBP and M-MBP level in the 3-TYP+HiLo group were significantly lower than the HiLo group(p < 0.01).(3)In terms of skeletal muscle vascular endothelial function and vascular density,ET-1 significantly decreased in the HiLo group(p < 0.05)and significantly higher in3-TYP group(p < 0.05).ET-1 was significantly higher in the 3-TYP + HiLo group than in the HiLo group(p < 0.05).NO levels and eNOS expression were significantly lower in the3-TYP+HiLo group than in the HiLo group(p < 0.01,p < 0.05).CD31 and VEGF expressions were lower in the 3-TYP+HiLo group than in the HiLo group(p < 0.05,p < 0.05).(4)In terms of mitochondrial quality control in skeletal muscle,the mitochondrial biosynthetic proteins,including the phosphorylation level of p38 MAPK,PGC-1α,COX Ⅳ,and Cytc expression were significantly lower in the 3-TYP+HiLo group than in the HiLo group(p < 0.05).The expression of mitophagy proteins PINK1 and Parkin in the 3-TYP+HiLo group was also significantly lower than that in the HiLo group(p< 0.01).The expression of mitochondrial fusion protein Opa1 and the mitochondrial fission proteins Fis/MFF were significantly lower in the 3-TYP+HiLo group than in the HiLo group(p < 0.05).Part Ⅲ: Effects of Sirt3 activation intervention on the changes in skeletal muscle microcirculation and mitochondrial quality control with the regulation of living high-training low.(1)In terms of Sirt3 downstream signals and the levels of oxidation and antioxidant of the skeletal muscle,Sirt3 and its downstream factors FOXO3 a and SOD2 expressions were significantly higher in the HiLo and the HNK groups than in the C group(p <0.05).CAT,GSH-Px,and SOD were also significantly higher in the C group(p < 0.05).The expression of Prx5 and Trx2 was elevated in the HiLo group(p < 0.05).Except for Sirt3,the changes in each of these indicators in the HNK+HiLo group were not significantly different from those in the HNK and the HiLo groups(p > 0.05).(2)In terms of skeletal muscle microcirculation,MBP and H-MBP were significantly higher in the HiLo and the HNK groups.The changes in each of these indicators in HNK+HiLo group were not different from those in HNK and HiLo group(p > 0.05).(3)In terms of skeletal muscle vascular endothelial function and vascular density,ET-1 levels was somewhat decreased in the HiLo group(p < 0.05).NO was elevated in the HiLo group(p < 0.05,p < 0.01).eNOS was elevated in the HiLo and the HNK groups(p < 0.05).The expression of CD31 and VEGF was higher in the HiLo and the HNK groups than in the C group(p < 0.05).No significant differences were observed among the HNK+HiLo,the HNK,and the HiLo group for all outcomes(p > 0.05),except that the NO in the HNK+HiLo group was significantly higher than in the other two groups.(4)In terms of mitochondrial quality control in skeletal muscle,the mitochondrial biosynthetic protein p-p38 MAPK levels significantly increased in the HiLo group(p <0.05)and significantly decreased in the HNK group(p < 0.01)compared to the C group.PGC-1α,COX Ⅳ and Cytc in the HiLo group and the HNK group were significantly higher in the C group(p < 0.05).The expression of mitophagy protein PINK1 and Parkin proteins significantly increased in the HiLo group and the HNK groups(p < 0.05,p < 0.01).The mitochondrial fusion protein Opa1 and the mitochondrial fission protein MFF significantly increased in the HiLo group and the HNK group(p < 0.05).Except for p-p38 MAPK,the changes in each of these indicators in the HNK+HiLo group were not different from those in the HNK and the HiLo groups(p > 0.05).Conclusions(1)HiLo interventions are more advantageous than other interventions in improving vascular endothelial diastolic capacity and modulating mitochondrial quality control in skeletal muscle.Sirt3/FOXO3 a signaling pathways in skeletal muscle are activated by living high-training low,which improves skeletal muscle antioxidant capacity.The change of microcirculatory function and the regulation of skeletal muscle mitochondrial quality control are enhanced in parallel after living high-training low training.(2)Sirt3 inhibition intervention partially attenuated the facilitation of microcirculatory perfusion levels in skeletal muscle under heated state by HiLo,which may be associated with changed vascular endothelial diastolic capacity.Sirt3 inhibition intervention also blocked the activation of the p38 MAPK/PGC-1α and PINK1/Parkin pathways by HiLo.This suggests that Sirt3 is involved in the regulation of skeletal muscle microcirculatory changes and mitochondrial quality control processes under conditions of HiLo training.(3)Activation of Sirt3 cause similar effects to HiLo intervention in enhancing microcirculation in skin and skeletal muscle,as well as partially similar effects in modulating mitochondrial quality control in skeletal muscle.In summary,this study demonstrated that Sirt3 may play an active role in the regulation of microcirculatory changes and mitochondrial quality control in skeletal muscle by HiLo training. |
