耐力训练及饮食限制影响心肌Pink1/Parkin线粒体自噬信号并提高自噬水平
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摘要
目的:观察耐力训练和饮食限制对小鼠心肌Pink1、Parkin及Drp-1的影响,探讨运动和饮食干预心肌Pink1/Parkin介导的线粒体自噬分子机制。方法:8周雄性C57BL/6小鼠随机分为安静对照组(C组)、耐力训练组(T组)、饮食限制组(D组)及耐力训练加饮食限制组(TD组),8只/组。C组不做运动,自由饮食;T组进行10周游泳训练,1次/d,5 d/周,第1周20 min/次,每周增加10 min,第8周维持在90 min/次直至实验结束;D组进行40%饮食削减;TD组进行耐力训练加饮食限制,方案分别与T、D组同。实验结束后取心肌,RT-PCR和Western blot技术检测Pink1、Parkin和Drp-1的mRNA及蛋白表达,透射电镜观察心肌细胞自噬发生情况。结果:1)与C组比较,T组体重下降幅度较小,心脏重量无显著变化(P>0.05),心系数显著升高(P<0.01);而D、TD组体重和心脏重量均显著下降(P<0.01)。2)T组心肌自噬体增多,仅有Pink1 mRNA和蛋白表达增高(P<0.05);D组心肌自噬体增加,仅有Drp-1蛋白表达增加(P<0.05);而TD组心肌自噬体增加最明显,同时线粒体受损严重,Parkin mRNA显著下降(P<0.05),但其蛋白无显著变化(P>0.05),而Pink1、Drp-1 mRNA及蛋白表达显著增加(P<0.05)。结论:耐力训练或饮食限制均能提高小鼠心肌自噬水平,耐力训练提高Pink1表达来增强Pink1/Parkin介导的线粒体自噬信号;饮食限制提高Drp-1蛋白表达,协助心肌线粒体自噬发生;耐力训练加饮食限制导致心肌线粒体等结构病变严重,Pink1和Drp-1蛋白表达提高是高水平线粒体自噬发生的机制。
Objective:This study investigated effects of endurance training and dietary restriction on myocardial Pink1,Parkin and Drp-1 expressions,and discussed molecular mechanism of Pink1/Parkin mediated mitophagy that was regulated by exercise and diet.Methods:Eight weeks old male C57BL/6 mice were randomly assigned to4 groups(n = 8) :control group(C group),endurance training group(T group),dietary restriction group(D group) and endurance training + dietary restriction group(TD group).T group undertook 10 weeks increased load swimming training,1 time/day,5 days/week.They swam 20 min in the 1st week,and increased 10 min per week and finished 90 min in the last 3 weeks.D group was suffered 40% food reduction.TD group undertook the same endurance training and dietary restriction.Cardiac muscle was collected after experiment,the mRNA and protein expressions of Pink1,Parkin and Drp-1 were tested by RT-PCR and Western blot,and the autophagy in myocardial cells was observed by transmission electron microscope.Results:1) Compared with C group,T group' body weight decreased less,heart weight had no difference(P > 0.05),heart coefficient increased significantly(P <0.01); D and TD groups' body weight and heart weight decreased(P < 0.01).2) Cardiac autophagosome of T group increased,and the mRNA and protein expressions of Pink1 increased(P < 0.05); cardiac autophagosomeof D group increased,and the protein expression of Drp-1 increased(P < 0.05); cardiac autophagosome of TD group increased the most,mitochondria was damaged seriously,and the mRNA expression of Parkin increased(P< 0.05),but Parkin protein expression didn't change(P > 0.05),the mRNA and protein expressions of Pink1 and Drp-1 increased(P < 0.05).Conclusions:Endurance training and dietary restriction can improve the autophagy level in mouse's heart; endurance training strengths the Pink1/Parkin mediated mitophagy signals in mitochondria by increasing Pink1 expression; dietary restriction improves Drp-1 protein expression,helps cardiac mitochondria autophagy; endurance training plus dietary restriction damage the structure of cardiac mitochondria,the improvement of Pink1 and Drp-1 protein expression is the mechanism of high level of mitochondrial autophagy.
Objective:This study investigated effects of endurance training and dietary restriction on myocardial Pink1,Parkin and Drp-1 expressions,and discussed molecular mechanism of Pink1/Parkin mediated mitophagy that was regulated by exercise and diet.Methods:Eight weeks old male C57BL/6 mice were randomly assigned to4 groups(n = 8) :control group(C group),endurance training group(T group),dietary restriction group(D group) and endurance training + dietary restriction group(TD group).T group undertook 10 weeks increased load swimming training,1 time/day,5 days/week.They swam 20 min in the 1st week,and increased 10 min per week and finished 90 min in the last 3 weeks.D group was suffered 40% food reduction.TD group undertook the same endurance training and dietary restriction.Cardiac muscle was collected after experiment,the mRNA and protein expressions of Pink1,Parkin and Drp-1 were tested by RT-PCR and Western blot,and the autophagy in myocardial cells was observed by transmission electron microscope.Results:1) Compared with C group,T group' body weight decreased less,heart weight had no difference(P > 0.05),heart coefficient increased significantly(P <0.01); D and TD groups' body weight and heart weight decreased(P < 0.01).2) Cardiac autophagosome of T group increased,and the mRNA and protein expressions of Pink1 increased(P < 0.05); cardiac autophagosomeof D group increased,and the protein expression of Drp-1 increased(P < 0.05); cardiac autophagosome of TD group increased the most,mitochondria was damaged seriously,and the mRNA expression of Parkin increased(P< 0.05),but Parkin protein expression didn't change(P > 0.05),the mRNA and protein expressions of Pink1 and Drp-1 increased(P < 0.05).Conclusions:Endurance training and dietary restriction can improve the autophagy level in mouse's heart; endurance training strengths the Pink1/Parkin mediated mitophagy signals in mitochondria by increasing Pink1 expression; dietary restriction improves Drp-1 protein expression,helps cardiac mitochondria autophagy; endurance training plus dietary restriction damage the structure of cardiac mitochondria,the improvement of Pink1 and Drp-1 protein expression is the mechanism of high level of mitochondrial autophagy.
引文
[1]Choi AM,Ryter SW,Levine B.Autophagy in human health and disease[J].N Engl J Med,2013,368(7):651-662.
[2]Quinsay MN,Thomas RL,Lee Y,et al.Bnip3-mediated mitochondrial autophagy is independent of the mitochondrial permeability transition pore[J].Autophagy,2010,6(7):855-862.
[3]Wei H,Liu L,Chen Q.Selective removal of mitochondria via mitophagy:distinct pathways for different mitochondrial stresses[J].Biochim Biophys Acta,2015,1853(10):2784-2790.
[4]谢凤,柳威,陈临溪.自噬参与心脏疾病调控的研究进展[J].生物化学与生物物理进展,2012,39(3):224-233.
[5]Thomas RL,Gustafsson AB.Mitochondrial autophagy:an essential quality control mechanism for myocardial homeostasis[J].Circ J,2013,77(10):2449-2454.
[6]Szafranski K,Mekhail K.The fine line between lifespan extension and shortening in response to caloric restriction[J].Nucleus,2014,5(1):56-65.
[7]马晓雯,常芸,王世强.长期耐力训练对大鼠心肌细胞自噬相关因子Beclin1和LC3的影响[J].体育科学,2016,36(2):66-70.
[8]Watanabe T,Takemura G,Kanamori H,et al.Restriction of food intake prevents postinfarction heart failure by enhancing autophagy in the surviving cardiomyocytes[J].Am J Pathol,2014,184(5):1384-1394.
[9]Figueira TR,Barros MH,Camargo AA,et al.Mitochondria as a source of reactive oxygen and nitrogen species:from molecular mechanisms to human health[J].Antioxid Redox Signal,2013,18(16):2029-2074.
[10]Figge MT,Osiewacz HD,Reichert AS.Quality control of mitochondria during aging:is there a good and a bad side of mitochondrial dynamics[J].Bioessays,2013,35(4):314-322.
[11]Baines CP.The cardiac mitochondrion:nexus of stress[J].Annu Rev Physiol,2010,72:61-80.
[12]Dorn GW 2nd,Kitsis RN.The mitochondrial dynamismmitophagy-cell death interactome:multiple roles performed by members of amitochondrial molecular ensemble[J].Circ Res,2015,116(1):167-182.
[13]Disatnik MH,Hwang S,Ferreira JC,et al.New therapeutics to modulate mitochondrial dynamics and mitophagy in cardiac diseases[J].J Mol Med(Berl),2015,93(3):279-287.
[14]Ding WX,Yin XM.Mitophagy:mechanisms,pathophysiological roles,and analysis[J].Biol Chem,2012,393(7):547-564.
[15]Pan W,Zhong Y,Cheng C,et al.MiR-30-regulated autophagy mediates angiotensin II-induced myocardial hypertrophy[J].PLo S One,2013,8(1):e53950.
[16]谢洪,李艳君,陈英玉.线粒体自噬[J].中国生物化学与分子生物学报,2011,27(12):1081-1087.
[17]Suen DF,Narendra DP,Tanaka A,et al.Parkin overexpression selects against a deleterious mt DNA mutation in heteroplasmic cybrid cells[J].Proc Natl Acad Sci,2010,107(26):11835-11840.
[18]Van Humbeeck C,Cornelissen T,Hofkens H,et al.Parkin interacts with Ambra1 to induce mitophagy[J].J Neurosci,2011,31(28):10249-10261.
[19]Kageyama Y,Hoshijima M,Seo K,et al.Parkin-independent mitophagy requires Drp1 and maintains the integrity of mammalian heart and brain[J].EMBO J,2014,33(23):2798-2813.
[20]马晓雯,常芸,王世强,等.不同强度不同时间耐力训练对于大鼠心肌细胞自噬发生程度的影响[J].中国运动医学杂志,2016,35(1):27-31.
[21]张建凯.饥饿对大鼠心肌自噬影响的实验研究[D].唐山:华北理工大学,2015.
[22]李丽,仝武军,樊荣,等.有氧运动通过上调心肌细胞自噬改善衰老心肌收缩舒张功能[J].心脏杂志,2014,26(1):29-34.
[23]张昊,王伟伟,薛过,等.运动训练通过诱导自噬、抑制凋亡改善老龄大鼠心脏功能[J].中国病理生理杂志,2014,30(1):11-17.
[24]贾绍辉,刘君,寇现娟,等.运动诱导的细胞自噬对小鼠心肌的保护作用[J].武汉体育学院学报,2014,48(10):53-56.
[25]Wohlgemuth SE,Julian D,Akin DE,et al.Autophagy in the heart and liver during normal aging and calorie restriction[J].Rejuvenation Res,2007,10(3):281-292.
[26]Chen Y,Dorn GW 2nd.PINK1-phosphorylated mitofusin2 is a Parkin receptor for culling damaged mitochondria[J].Science,2013,340(6131):471-475.
[27]López-Lluch G,Hunt N,Jones B,et al.Calorie restriction induces mitochondrial biogenesis and bioenergetic efficiency[J].Proc Natl Acad Sci USA,2006,103(6):1768-1773.
[28]Khraiwesh H,López-Domínguez JA,López-Lluch G,et al.Alterations of ultrastructural and fission/fusion markers in hepatocyte mitochondria from mice following calorierestriction with different dietary fats[J].J Gerontol A Biol Sci Med Sci,2013,68(9):1023-1034.
[29]刘垒,冯杜,朱玉山,等.线粒体自噬的研究进展[J].中国细胞生物学学报,2012,34(10):959-966.
[30]Watanabe T,Saotome M,Nobuhara M,et al.Roles of mitochondrial fragmentation and reactive oxygen species in mitochondrial dysfunction and myocardial insulin resistance[J].Exp Cell Res,2014,323(2):314-325.
[2]Quinsay MN,Thomas RL,Lee Y,et al.Bnip3-mediated mitochondrial autophagy is independent of the mitochondrial permeability transition pore[J].Autophagy,2010,6(7):855-862.
[3]Wei H,Liu L,Chen Q.Selective removal of mitochondria via mitophagy:distinct pathways for different mitochondrial stresses[J].Biochim Biophys Acta,2015,1853(10):2784-2790.
[4]谢凤,柳威,陈临溪.自噬参与心脏疾病调控的研究进展[J].生物化学与生物物理进展,2012,39(3):224-233.
[5]Thomas RL,Gustafsson AB.Mitochondrial autophagy:an essential quality control mechanism for myocardial homeostasis[J].Circ J,2013,77(10):2449-2454.
[6]Szafranski K,Mekhail K.The fine line between lifespan extension and shortening in response to caloric restriction[J].Nucleus,2014,5(1):56-65.
[7]马晓雯,常芸,王世强.长期耐力训练对大鼠心肌细胞自噬相关因子Beclin1和LC3的影响[J].体育科学,2016,36(2):66-70.
[8]Watanabe T,Takemura G,Kanamori H,et al.Restriction of food intake prevents postinfarction heart failure by enhancing autophagy in the surviving cardiomyocytes[J].Am J Pathol,2014,184(5):1384-1394.
[9]Figueira TR,Barros MH,Camargo AA,et al.Mitochondria as a source of reactive oxygen and nitrogen species:from molecular mechanisms to human health[J].Antioxid Redox Signal,2013,18(16):2029-2074.
[10]Figge MT,Osiewacz HD,Reichert AS.Quality control of mitochondria during aging:is there a good and a bad side of mitochondrial dynamics[J].Bioessays,2013,35(4):314-322.
[11]Baines CP.The cardiac mitochondrion:nexus of stress[J].Annu Rev Physiol,2010,72:61-80.
[12]Dorn GW 2nd,Kitsis RN.The mitochondrial dynamismmitophagy-cell death interactome:multiple roles performed by members of amitochondrial molecular ensemble[J].Circ Res,2015,116(1):167-182.
[13]Disatnik MH,Hwang S,Ferreira JC,et al.New therapeutics to modulate mitochondrial dynamics and mitophagy in cardiac diseases[J].J Mol Med(Berl),2015,93(3):279-287.
[14]Ding WX,Yin XM.Mitophagy:mechanisms,pathophysiological roles,and analysis[J].Biol Chem,2012,393(7):547-564.
[15]Pan W,Zhong Y,Cheng C,et al.MiR-30-regulated autophagy mediates angiotensin II-induced myocardial hypertrophy[J].PLo S One,2013,8(1):e53950.
[16]谢洪,李艳君,陈英玉.线粒体自噬[J].中国生物化学与分子生物学报,2011,27(12):1081-1087.
[17]Suen DF,Narendra DP,Tanaka A,et al.Parkin overexpression selects against a deleterious mt DNA mutation in heteroplasmic cybrid cells[J].Proc Natl Acad Sci,2010,107(26):11835-11840.
[18]Van Humbeeck C,Cornelissen T,Hofkens H,et al.Parkin interacts with Ambra1 to induce mitophagy[J].J Neurosci,2011,31(28):10249-10261.
[19]Kageyama Y,Hoshijima M,Seo K,et al.Parkin-independent mitophagy requires Drp1 and maintains the integrity of mammalian heart and brain[J].EMBO J,2014,33(23):2798-2813.
[20]马晓雯,常芸,王世强,等.不同强度不同时间耐力训练对于大鼠心肌细胞自噬发生程度的影响[J].中国运动医学杂志,2016,35(1):27-31.
[21]张建凯.饥饿对大鼠心肌自噬影响的实验研究[D].唐山:华北理工大学,2015.
[22]李丽,仝武军,樊荣,等.有氧运动通过上调心肌细胞自噬改善衰老心肌收缩舒张功能[J].心脏杂志,2014,26(1):29-34.
[23]张昊,王伟伟,薛过,等.运动训练通过诱导自噬、抑制凋亡改善老龄大鼠心脏功能[J].中国病理生理杂志,2014,30(1):11-17.
[24]贾绍辉,刘君,寇现娟,等.运动诱导的细胞自噬对小鼠心肌的保护作用[J].武汉体育学院学报,2014,48(10):53-56.
[25]Wohlgemuth SE,Julian D,Akin DE,et al.Autophagy in the heart and liver during normal aging and calorie restriction[J].Rejuvenation Res,2007,10(3):281-292.
[26]Chen Y,Dorn GW 2nd.PINK1-phosphorylated mitofusin2 is a Parkin receptor for culling damaged mitochondria[J].Science,2013,340(6131):471-475.
[27]López-Lluch G,Hunt N,Jones B,et al.Calorie restriction induces mitochondrial biogenesis and bioenergetic efficiency[J].Proc Natl Acad Sci USA,2006,103(6):1768-1773.
[28]Khraiwesh H,López-Domínguez JA,López-Lluch G,et al.Alterations of ultrastructural and fission/fusion markers in hepatocyte mitochondria from mice following calorierestriction with different dietary fats[J].J Gerontol A Biol Sci Med Sci,2013,68(9):1023-1034.
[29]刘垒,冯杜,朱玉山,等.线粒体自噬的研究进展[J].中国细胞生物学学报,2012,34(10):959-966.
[30]Watanabe T,Saotome M,Nobuhara M,et al.Roles of mitochondrial fragmentation and reactive oxygen species in mitochondrial dysfunction and myocardial insulin resistance[J].Exp Cell Res,2014,323(2):314-325.