线粒体分裂蛋白1在安静状态下和力竭运动中对骨骼肌线粒体质量的调控作用

Mitochondrial Fission 1 Protein Regulates Mitochondrial Quality of Skeletal Muscle in Resting State and Exhaustive Exercise

目的:探索骨骼肌线粒体分裂蛋白1(mitochondrial fission 1 protein,Fis1)在哺乳动物安静状态和力竭运动应激下,对不同类型骨骼肌线粒体质量的调控作用,进而阐明骨骼肌Fis1对运动能力的影响及其可能机制。方法:运用LoxP/Cre技术建立骨骼肌特异性敲除Fis1的小鼠模型(Fis1KO)及其对照组(WT),通过PCR鉴定小鼠基因型和Western-blot检测Fis1的蛋白表达以确认模型的成功建立;随后将实验小鼠分为4组,即安静对照组(WT)、骨骼肌特异性敲除Fis1组(Fis1KO)、力竭运动组(WT EEE)和骨骼肌特异性敲除Fis1结合力竭运动组(Fis1KO EEE),n=10~11;WT EEE和Fis1KO EEE这2组小鼠进行递增负荷至力竭的跑台运动(endurance exhaustive exercise,EEE)并记录力竭时间;分别使用GOMORI染色和电子显微镜检测缺失Fis1对小鼠骨骼肌线粒体密度和形态的影响;使用相关试剂盒和酶标仪检测线粒体氧化磷酸化(oxidative phosphorylation,OXPHOS)复合体(Complex)和柠檬酸合酶(Citrate Synthase,CS)的活性。结果:1)通过PCR鉴定基因型并筛选出对照组和基因敲除小鼠。Western-blot结果显示,敲除Fis1的比目鱼肌和腓肠肌内均无Fis1的表达。2)GOMORI染色结果显示与WT相比,Fis1KO比目鱼肌肌纤维内线粒体稀疏,着色变浅;不同组别小鼠的腓肠肌GOMORI染色结果并未出现明显差异。3)电镜结果显示丢失Fis1使得比目鱼肌(慢肌)内线粒体面积增大(过度融合,P<0.05);而在力竭运动后,Fis1KO EEE组小鼠的比目鱼肌肌浆网明显肿胀。4)电镜结果显示敲除Fis1使得腓肠肌(混合肌)出现了肿胀线粒体,而在力竭运动后,Fis1KO EEE组小鼠的腓肠肌的终池极度肿胀。5)安静状态下WT与Fis1KO这2组小鼠骨骼肌线粒体功能无显著差异,但与运动前Fis1KO组相比,Fis1KO EEE组的比目鱼肌Complex Ⅳ的活性显著降低(P<0.001),而与运动前WT组相比,WT EEE组比目鱼肌Complex Ⅳ的活性仅轻微下降(P<0.05);同样是与运动前Fis1KO组相比,Fis1KO EEE组的腓肠肌CS(P<0.001)、Complex Ⅰ(P<0.01)和Complex Ⅳ(P<0.01)的活性均显著下降,而与运动前WT组相比,WT EEE组小鼠的腓肠肌内仅有CS的活性降低(P<0.01)。6)Fis1KO组小鼠的力竭运动时间较WT组明显减少(P<0.05)。结论:在安静状态下,Fis1在慢肌内参与线粒体分裂并维持线粒体密度,在混合肌中则维持正常的线粒体形态避免出现肿胀的线粒体;Fis1在力竭运动中维持骨骼肌内质网的正常形态并保护骨骼肌线粒体功能免于受损,进而维持小鼠的运动能力。

Objective:This study was conducted to explore the function of Fis1 in regulating mitochondrial quality in mammalian different skeletal muscles under resting state and exhaustive exercise stress,so as to clarify the effect and molecular mechanisms of skeletal muscle Fis1 on aerobic exercise capacity.Methods:The mouse model of specific knockout Fis1 in skeletal muscle(Fis1KO)and control group(WT)were generated through LoxP/Cre technique.The genotype of the mice and Fis1 protein level was detected by PCR and Western-blot respectively.Mice were then divided into 4 groups:WT,Fis1KO,WT EEE,Fis1KO EEE,n=10-11.Mice from WT EEE and Fis1KO EEE groups were forced to run until exhausted,the exhaustion time was recorded.GOMORI staining and electron microscopy were used to observe mitochondrial density and ultrastructure respectively.We also assessed the activity of mitochondrial oxidative phosphorylation(OXPHOS)complexes and CS through related kits and microplate reader before and after exhaustive exercise.Results:1)We confirmed Fis1 loss in skeletal muscle through PCR and Western-blot;2)In soleus(slow muscle),loss of Fis1 shallowed the staining of mitochondria;3)GOMORI staining appeared largely unchanged in gastrocnemius from different groups;4)Knocking out Fis1 enlarged the mitochondrial area(hyper-fusion)(P<0.05)of soleus at resting state.The swollen sarcoplasmic reticulum was found in soleus from Fis1 KO EEE group;5)Swollen mitochondria were found in Fis1KO gastrocnemius(mixed muscle)at resting state,and swollen terminal cisterna(TC)was observed in gastrocnemius from Fis1 KO EEE group;6)There is no significant change in activity of mitochondrial OXPHOS Complexes and CS in skeletal muscle of either genotype before exhaustive exercise.However,the activity of Complex Ⅳ of soleus from Fis1KO EEE was significantly reduced(P<0.001)compared with that of Fis1KO group,while the Complex Ⅳ activity of soleus from WT EEE group only showed a mild decrease(P<0.05)compared with WT group.Similar with soleus,the activity of citrate synthase(CS)(P<0.001),Complex I(P<0.01),Complex Ⅳ(P<0.01)of gastrocnemius from Fis1KO EEE group showed a significant decrease compared with Fis1KO group,but only CS activity(P<0.01)decreased in gastrocnemius from WT EEE group compared with that of WT;7)The exhaustive running time of Fis1KO group was significantly lower than that of WT group(P<0.05).Conclusion:At resting state,Fis1 mediates mitochondrial fission and maintains mitochondrial density in slow muscle,as well as protects mitochondrial morphology in mixed muscle from swollen mitochondria appearing.During exhaustive exercise,skeletal muscle Fis1 maintains endoplasmic reticulum morphology and protects mitochondrial function from damage,so as to plays key role on aerobic capacity of mice.