低氧调控EIMD后calpain活性与肌细胞膜损伤机制的探索研究
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摘要
目的:观察低氧对运动性肌损伤(exercise-induced muscle damage,EIMD)后肌细胞膜损伤、钙激活中性蛋白酶(calpain)和肌细胞膜骨架蛋白(dystrophin、utrophin和dysferlin)的影响,探索低氧调控EIMD后肌细胞膜损伤的可能机制。方法:110只雄性SD健康大鼠分两批进行实验。研究Ⅰ:70只大鼠随机均分为安静对照组、常氧24 h、48 h和72 h组以及低氧24 h、48 h和72 h组,每组10只。大鼠在-16°跑台上以26.8 m/min速度持续运动5 min,运动10次,组间休息1 min。低氧和常氧各组大鼠运动后注射伊文氏蓝荧光染色剂观察膜损伤情况,用RT-q PCR和Western blot测定腓肠肌calpain和膜骨架蛋白的mRNA表达和蛋白含量。研究Ⅱ:40只大鼠随机均分为空白对照组、安慰剂24 h、48 h组和calpain抑制剂24 h、48 h组,每组8只。测定calpain抑制剂对其蛋白含量的影响,观察膜损伤情况,并验证calpain在EIMD后低氧损伤肌细胞膜中所起的作用。结果:大鼠EIMD后,1)低氧各组膜的通透性增加、完整性被破坏,阳性细胞率(positive ratio of cell,PRC)与对照组和常氧对应组相比有显著差异(P<0.01);2)低氧72 h组calpain1、calpain2的mRNA表达显著高于常氧72 h组(P<0.05),低氧各组calpain1、calpain2和calpain3的蛋白含量与常氧对应组相比无显著差异(P>0.05);3)低氧24 h组dystrophin、utrophin的m RNA表达显著低于常氧24 h组(P<0.01),但两组的蛋白含量无显著差异(P>0.05)。低氧各组dys-ferlin的mRNA表达和蛋白含量与常氧各组相比无显著差异(P> 0.05)。4)抑制calpain1、calpain2活性后,低氧24 h组calpain蛋白含量显著降低(P<0.05),低氧各组阳性细胞率显著低于安慰剂各组(P<0.01),但仍显著高于对照组(P<0.05)。结论:1) calpain1、calpain2被激活与低氧加剧大鼠EIMD后的肌细胞膜损伤有关,但calpain并非通过降解膜骨架蛋白dystrophin、utrophin和dysferlin的途径而导致膜损伤。2)抑制calpain1、calpain2活性能有效减轻但不能避免低氧加剧EIMD后的膜损伤,其中还可能存在其它的调节机制。
Objective: To observe the effects of hypoxia on sarcolemma damage, calpain activity and membrane cytoskeleton proteins after exercise-induced muscle damage(EIMD), and to explore the possible mechanism of hypoxia regulates sarcolemma damage after EIMD. Methods:One hundred ten healthy male SD rats were tested in two studies. Study Ⅰ: Seventy rats were randomly divided into control, normoxia(N) 24, 48 and 72 h group and hypoxia(H) 24, 48 and72 h groups. Rats performed downhill run at 26.8 m/min in-16° treadmill(continuous 5 min/set, intermittent interval 1 min, 10 sets). Observing sarcolemma injury by injecting Evan's Blue Dye after downhill run in hypoxia and normoxia groups, and investigates mRNA and protein content of calpain and membrane cytoskeleton by RT-qPCR and Western blot method. Study Ⅱ:Forty rats were randomly divided into blank, placebo(Pl-) 24 h, 48 h and calpain inhibitor(I-)24 h, 48 h groups. To investigate changes of calpain inhibitor on the protein content of calpain and observe sarcolemma injury extent, and con?rm the role of calpain on hypoxia induced sarcolemma injury after EIMD. Results: After EIMD, 1) hypoxia group's membrane permeability was increased and the integrity was decreased. There were signi?cant differences of positive ratio of cell(PRC) in hypoxia groups compared with control and normal groups(P< 0.01). 2) The mRNA of calpain1 and calpain2 in the H72 group was signi?cantly higher than N72 group(P<0.05). There was no signi?cant difference in protein content of calpain1, calpain2 and calpain3 between hypoxia and normoxia groups(P> 0.05). 3) The mRNA of dystrophin and utrophin in H24 group was signi?cantly lower than N24 group(P<0.01), but no signi?cant difference in protein content between the two groups(P> 0.05). The mRNA and protein content of dysferlin in hypoxia group were not signi?cantly different from normoxia group(P>0.05). 4) After injecting inhibitor, the protein content of calpain1 and calpain2 signi?cantly decreased(P< 0.05).The PRC in hypoxia groups was significantly lower than placebo groups(P< 0.01), and was significantly higher than control group(P< 0.05). Conclusion: 1) Activation of calpain1 and calpain2 are involved in hypoxia exacerbation sarcolemma injury after EIMD, but not through degradation membrane cytoskeleton protein(dystrophin, utrophin and dysferlin). 2) Inhibition of calpain1, calpain2 activity can effectively reduce but can not avoid sarcolemma injury exacerbated by hypoxia after EIMD. There may be other related regulatory mechanisms in calpain way.
Objective: To observe the effects of hypoxia on sarcolemma damage, calpain activity and membrane cytoskeleton proteins after exercise-induced muscle damage(EIMD), and to explore the possible mechanism of hypoxia regulates sarcolemma damage after EIMD. Methods:One hundred ten healthy male SD rats were tested in two studies. Study Ⅰ: Seventy rats were randomly divided into control, normoxia(N) 24, 48 and 72 h group and hypoxia(H) 24, 48 and72 h groups. Rats performed downhill run at 26.8 m/min in-16° treadmill(continuous 5 min/set, intermittent interval 1 min, 10 sets). Observing sarcolemma injury by injecting Evan's Blue Dye after downhill run in hypoxia and normoxia groups, and investigates mRNA and protein content of calpain and membrane cytoskeleton by RT-qPCR and Western blot method. Study Ⅱ:Forty rats were randomly divided into blank, placebo(Pl-) 24 h, 48 h and calpain inhibitor(I-)24 h, 48 h groups. To investigate changes of calpain inhibitor on the protein content of calpain and observe sarcolemma injury extent, and con?rm the role of calpain on hypoxia induced sarcolemma injury after EIMD. Results: After EIMD, 1) hypoxia group's membrane permeability was increased and the integrity was decreased. There were signi?cant differences of positive ratio of cell(PRC) in hypoxia groups compared with control and normal groups(P< 0.01). 2) The mRNA of calpain1 and calpain2 in the H72 group was signi?cantly higher than N72 group(P<0.05). There was no signi?cant difference in protein content of calpain1, calpain2 and calpain3 between hypoxia and normoxia groups(P> 0.05). 3) The mRNA of dystrophin and utrophin in H24 group was signi?cantly lower than N24 group(P<0.01), but no signi?cant difference in protein content between the two groups(P> 0.05). The mRNA and protein content of dysferlin in hypoxia group were not signi?cantly different from normoxia group(P>0.05). 4) After injecting inhibitor, the protein content of calpain1 and calpain2 signi?cantly decreased(P< 0.05).The PRC in hypoxia groups was significantly lower than placebo groups(P< 0.01), and was significantly higher than control group(P< 0.05). Conclusion: 1) Activation of calpain1 and calpain2 are involved in hypoxia exacerbation sarcolemma injury after EIMD, but not through degradation membrane cytoskeleton protein(dystrophin, utrophin and dysferlin). 2) Inhibition of calpain1, calpain2 activity can effectively reduce but can not avoid sarcolemma injury exacerbated by hypoxia after EIMD. There may be other related regulatory mechanisms in calpain way.
引文
金其贯,刘霞,李淑艳,等,2010.反复离心运动对大鼠骨骼肌损伤和蛋白降解机制的影响[J].体育科学,30(6),76-80.
金其贯,刘霞,李淑艳,等,2011.急性离心运动后骨骼肌超微结构、钙依赖性蛋白酶和泛素的动态变化[J].中国运动医学杂志,30(8),724-728.
王金发,2003.细胞生物学[M].北京:科学出版社:70-71.
徐飞,黄巧婷,曹建民,等,2017.低氧调控EIMD后肌细胞膜损伤MAPK机制的探索与验证[J].体育科学,37(11),63-71.
ADAMS B,MAPANGA R F,ESSOP M F,2015.Partial inhibition of the ubiquitin-proteasome system ameliorates cardiac dysfunction following ischemia-reperfusion in the presence of high glucose[J].Cardiovasc Diabetol,14,94.
ARMSTRONG R B,OGILVIE R W,SCHWANE J A,1983.Eccentric exercise-induced injury to rat skeletal muscle[J].J Appl Physiol Respir Environ Exerc Physiol,54(1),80-93.
BANSAL D,MIYAKE K,VOGEL S S,et al.,2003.Defective membrane repair in dysferlin-deficient muscular dystrophy[J].Nature,423(6936),168-172.
BARTOLI M,RICHARD I,2005.Calpains in muscle wasting[J].Int J Biochem Cell Biol,37(10),2115-2133.
CARDENAS A M,GONZALEZ-JAMETT A M,CEA L A,et al.,2016.Dysferlin function in skeletal muscle:Possible pathological mechanisms and therapeutical targets in dysferlinopathies[J].Exp Neurol,283(Pt A),246-254.
CULLY T R.,MURPHY R M,ROBERTS L,et al.,2017.Human skeletal muscle plasmalemma alters its structure to change its Ca2+-handling following heavy-load resistance exercise[J].Nat Commun,8,14266.
D’HULST G,JAMART C,VAN THIENEN R,et al.,2013.Effect of acute environmental hypoxia on protein metabolism in human skeletal muscle[J].Acta Physiol(Oxf),208(3),251-264.
FEASSON L,STOCKHOLM D,FREYSSENET D,et al.,2002.Molecular adaptations of neuromuscular disease-associated proteins in response to eccentric exercise in human skeletal muscle[J].J Physiol,543(Pt 1),297-306.
FREITAS A C,FIGUEIREDO M J,CAMPOS E C,et al.,2016.Activation of Both the Calpain and Ubiquitin-Proteasome Systems Contributes to Septic Cardiomyopathy through Dystrophin Loss/Disruption and mTOR Inhibition[J].PLoS One,11(11),e0166839.
GUIRAUD S,EDWARDS B,SQUIRE S E,et al.,2017.Identification of serum protein biomarkers for utrophin based DMD therapy[J].Sci Rep,7,43697.
HAMER P W,MCGEACHIE J M,DAVIES M J,et al.,2002.Evans Blue Dye as an in vivo marker of myofibre damage:optimising parameters for detecting initial myofibre membrane permeability[J].JAnat,200(Pt 1),69-79.
HASENOEHRL T,WESSNER B,TSCHAN H,et al.,2017.Eccentric resistance training intensity may affect the severity of exercise induced muscle damage[J].J Sports Med Phys Fitness,57(9),1195-1204.
HIRATA M,SHEARER T R,AZUMA M,2015.Hypoxia Activates Calpains in the Nerve Fiber Layer of Monkey Retinal Explants[J].Invest Ophthalmol Vis Sci,56(10),6049-6057.
JONES S W,HILL R J,KRASNEY P A,et al.,2004.Disuse atrophy and exercise rehabilitation in humans profoundly affects the expression of genes associated with the regulation of skeletal muscle mass[J].FASEB J,18(9),1025-1027.
JOUNG H,EOM G H,CHOE N,et al.,2014.Ret finger protein mediates Pax7-induced ubiquitination of MyoD in skeletal muscle atrophy[J].Cell Signal,26(10),2240-2248.
KANZAKI K,WATANABE D,KURATANI M,et al.,2017.Role of calpain in eccentric contraction-induced proteolysis of Ca2+-regulatory proteins and force depression in rat fast-twitch skeletal muscle[J].J Appl Physiol(1985),122(2),396-405.
KOVACS L,HAN W,RAFIKOV R,et al.,2016.Activation of Calpain-2by Mediators in Pulmonary Vascular Remodeling of Pulmonary Arterial Hypertension[J].Am J Respir Cell Mol Biol,54(3),384-393.
LIEBER R L,THORNELL L E,FRIDEN J,1996.Muscle cytoskeletal disruption occurs within the first 15 min of cyclic eccentric contraction[J].J Appl Physiol(1985),80(1),278-284.
LOMONOSOVA Y N,SHENKMAN B S,KALAMKAROV G R,et al.,2014.L-arginine supplementation protects exercise performance and structural integrity of muscle fibers after a single bout of eccentric exercise in rats[J].PLoS One,9(4),e94448.
LUO F,SHI J,SHI Q,et al.,2016.Mitogen-Activated Protein Kinases and Hypoxic/Ischemic Nephropathy[J].Cell Physiol Biochem,39(3),1051-1067.
MANCINELLI R,DI FILIPPO E S,VERRATTI V,et al.,2016.The Regenerative Potential of Female Skeletal Muscle upon Hypobaric Hypoxic Exposure[J].Front Physiol,7,303
MARTIN N R,AGUILAR-AGON K,ROBINSON G P,et al.,2017.Hypoxia Impairs Muscle Function and Reduces Myotube Size in Tissue Engineered Skeletal Muscle[J].J Cell Biochem,118(9),2599-2605.
MURPHY R M,LAMB G D,2009.Endogenous calpain-3 activation is primarily governed by small increases in resting cytoplasmic[Ca2+]and is not dependent on stretch[J].J Biol Chem,284(12),7811-7819.
MURPHY R M,SNOW R J,LAMB G D,2006.mu-Calpain and calpain-3 are not autolyzed with exhaustive exercise in humans[J].Am J Physiol Cell Physiol,290(1),C116-122.
MURPHY R M,VISSING K,LATCHMAN H,et al.,2011.Activation of skeletal muscle calpain-3 by eccentric exercise in humans does not result in its translocation to the nucleus or cytosol[J].J Appl Physiol(1985),111(5),1448-1458.
NOSAKA K,CLARKSON P M,1995.Muscle damage following repeated bouts of high force eccentric exercise[J].Med Sci Sports Exerc,27(9),1263-1269.
ROUDAUT C,LE ROY F,SUEL L,et al.,2013.Restriction of calpain3 expression to the skeletal muscle prevents cardiac toxicity and corrects pathology in a murine model of limb-girdle muscular dystrophy[J].Circulation,128(10),1094-1104.
ROVIDA E,DI MAIRA G,TUSA I,et al.,2015.The mitogen-activated protein kinase ERK5 regulates the development and growth of hepatocellular carcinoma[J].Gut,64(9),1454-1465.
SPENCER M J,GUYON J R,SORIMACHI H,et al.,2002.Stable expression of calpain 3 from a muscle transgene in vivo:immature muscle in transgenic mice suggests a role for calpain 3 in muscle maturation[J].Proc Natl Acad Sci U S A,99(13),8874-8879.
STELTER Z,STRAKOVA J,YELLAMILLI A,et al.,2016.Hypoxia-induced cardiac injury in dystrophic mice[J].Am J Physiol Heart Circ Physiol,310(7),H938-948.
WANG N,KANG H S,AHMMED G,et al.,2016.Calpain activation by ROS mediates human ether-a-go-go-related gene protein degradation by intermittent hypoxia[J].Am J Physiol Cell Physiol,310(5),C329-336.
(1)各组如有大鼠脱落,用备用大鼠补齐样本。
(2)24 h组伊文氏蓝注射时间点为运动后即刻,48 h组伊文氏蓝在运动后即刻和运动后24 h共注射2次。
(1)本研究N24组calpain3的mRNA表达显著高于C组(图3C),与Murphy等(2011)发现离心运动短期内激活calpain3的结果一致,说明大强度离心运动能够激活calpain3,但这不是本研究要证明和关注的内容,故并未展开讨论。
(2)是一个巨大蛋白,由3685个氨基酸组成,分子量约为427 kD。
金其贯,刘霞,李淑艳,等,2011.急性离心运动后骨骼肌超微结构、钙依赖性蛋白酶和泛素的动态变化[J].中国运动医学杂志,30(8),724-728.
王金发,2003.细胞生物学[M].北京:科学出版社:70-71.
徐飞,黄巧婷,曹建民,等,2017.低氧调控EIMD后肌细胞膜损伤MAPK机制的探索与验证[J].体育科学,37(11),63-71.
ADAMS B,MAPANGA R F,ESSOP M F,2015.Partial inhibition of the ubiquitin-proteasome system ameliorates cardiac dysfunction following ischemia-reperfusion in the presence of high glucose[J].Cardiovasc Diabetol,14,94.
ARMSTRONG R B,OGILVIE R W,SCHWANE J A,1983.Eccentric exercise-induced injury to rat skeletal muscle[J].J Appl Physiol Respir Environ Exerc Physiol,54(1),80-93.
BANSAL D,MIYAKE K,VOGEL S S,et al.,2003.Defective membrane repair in dysferlin-deficient muscular dystrophy[J].Nature,423(6936),168-172.
BARTOLI M,RICHARD I,2005.Calpains in muscle wasting[J].Int J Biochem Cell Biol,37(10),2115-2133.
CARDENAS A M,GONZALEZ-JAMETT A M,CEA L A,et al.,2016.Dysferlin function in skeletal muscle:Possible pathological mechanisms and therapeutical targets in dysferlinopathies[J].Exp Neurol,283(Pt A),246-254.
CULLY T R.,MURPHY R M,ROBERTS L,et al.,2017.Human skeletal muscle plasmalemma alters its structure to change its Ca2+-handling following heavy-load resistance exercise[J].Nat Commun,8,14266.
D’HULST G,JAMART C,VAN THIENEN R,et al.,2013.Effect of acute environmental hypoxia on protein metabolism in human skeletal muscle[J].Acta Physiol(Oxf),208(3),251-264.
FEASSON L,STOCKHOLM D,FREYSSENET D,et al.,2002.Molecular adaptations of neuromuscular disease-associated proteins in response to eccentric exercise in human skeletal muscle[J].J Physiol,543(Pt 1),297-306.
FREITAS A C,FIGUEIREDO M J,CAMPOS E C,et al.,2016.Activation of Both the Calpain and Ubiquitin-Proteasome Systems Contributes to Septic Cardiomyopathy through Dystrophin Loss/Disruption and mTOR Inhibition[J].PLoS One,11(11),e0166839.
GUIRAUD S,EDWARDS B,SQUIRE S E,et al.,2017.Identification of serum protein biomarkers for utrophin based DMD therapy[J].Sci Rep,7,43697.
HAMER P W,MCGEACHIE J M,DAVIES M J,et al.,2002.Evans Blue Dye as an in vivo marker of myofibre damage:optimising parameters for detecting initial myofibre membrane permeability[J].JAnat,200(Pt 1),69-79.
HASENOEHRL T,WESSNER B,TSCHAN H,et al.,2017.Eccentric resistance training intensity may affect the severity of exercise induced muscle damage[J].J Sports Med Phys Fitness,57(9),1195-1204.
HIRATA M,SHEARER T R,AZUMA M,2015.Hypoxia Activates Calpains in the Nerve Fiber Layer of Monkey Retinal Explants[J].Invest Ophthalmol Vis Sci,56(10),6049-6057.
JONES S W,HILL R J,KRASNEY P A,et al.,2004.Disuse atrophy and exercise rehabilitation in humans profoundly affects the expression of genes associated with the regulation of skeletal muscle mass[J].FASEB J,18(9),1025-1027.
JOUNG H,EOM G H,CHOE N,et al.,2014.Ret finger protein mediates Pax7-induced ubiquitination of MyoD in skeletal muscle atrophy[J].Cell Signal,26(10),2240-2248.
KANZAKI K,WATANABE D,KURATANI M,et al.,2017.Role of calpain in eccentric contraction-induced proteolysis of Ca2+-regulatory proteins and force depression in rat fast-twitch skeletal muscle[J].J Appl Physiol(1985),122(2),396-405.
KOVACS L,HAN W,RAFIKOV R,et al.,2016.Activation of Calpain-2by Mediators in Pulmonary Vascular Remodeling of Pulmonary Arterial Hypertension[J].Am J Respir Cell Mol Biol,54(3),384-393.
LIEBER R L,THORNELL L E,FRIDEN J,1996.Muscle cytoskeletal disruption occurs within the first 15 min of cyclic eccentric contraction[J].J Appl Physiol(1985),80(1),278-284.
LOMONOSOVA Y N,SHENKMAN B S,KALAMKAROV G R,et al.,2014.L-arginine supplementation protects exercise performance and structural integrity of muscle fibers after a single bout of eccentric exercise in rats[J].PLoS One,9(4),e94448.
LUO F,SHI J,SHI Q,et al.,2016.Mitogen-Activated Protein Kinases and Hypoxic/Ischemic Nephropathy[J].Cell Physiol Biochem,39(3),1051-1067.
MANCINELLI R,DI FILIPPO E S,VERRATTI V,et al.,2016.The Regenerative Potential of Female Skeletal Muscle upon Hypobaric Hypoxic Exposure[J].Front Physiol,7,303
MARTIN N R,AGUILAR-AGON K,ROBINSON G P,et al.,2017.Hypoxia Impairs Muscle Function and Reduces Myotube Size in Tissue Engineered Skeletal Muscle[J].J Cell Biochem,118(9),2599-2605.
MURPHY R M,LAMB G D,2009.Endogenous calpain-3 activation is primarily governed by small increases in resting cytoplasmic[Ca2+]and is not dependent on stretch[J].J Biol Chem,284(12),7811-7819.
MURPHY R M,SNOW R J,LAMB G D,2006.mu-Calpain and calpain-3 are not autolyzed with exhaustive exercise in humans[J].Am J Physiol Cell Physiol,290(1),C116-122.
MURPHY R M,VISSING K,LATCHMAN H,et al.,2011.Activation of skeletal muscle calpain-3 by eccentric exercise in humans does not result in its translocation to the nucleus or cytosol[J].J Appl Physiol(1985),111(5),1448-1458.
NOSAKA K,CLARKSON P M,1995.Muscle damage following repeated bouts of high force eccentric exercise[J].Med Sci Sports Exerc,27(9),1263-1269.
ROUDAUT C,LE ROY F,SUEL L,et al.,2013.Restriction of calpain3 expression to the skeletal muscle prevents cardiac toxicity and corrects pathology in a murine model of limb-girdle muscular dystrophy[J].Circulation,128(10),1094-1104.
ROVIDA E,DI MAIRA G,TUSA I,et al.,2015.The mitogen-activated protein kinase ERK5 regulates the development and growth of hepatocellular carcinoma[J].Gut,64(9),1454-1465.
SPENCER M J,GUYON J R,SORIMACHI H,et al.,2002.Stable expression of calpain 3 from a muscle transgene in vivo:immature muscle in transgenic mice suggests a role for calpain 3 in muscle maturation[J].Proc Natl Acad Sci U S A,99(13),8874-8879.
STELTER Z,STRAKOVA J,YELLAMILLI A,et al.,2016.Hypoxia-induced cardiac injury in dystrophic mice[J].Am J Physiol Heart Circ Physiol,310(7),H938-948.
WANG N,KANG H S,AHMMED G,et al.,2016.Calpain activation by ROS mediates human ether-a-go-go-related gene protein degradation by intermittent hypoxia[J].Am J Physiol Cell Physiol,310(5),C329-336.
(1)各组如有大鼠脱落,用备用大鼠补齐样本。
(2)24 h组伊文氏蓝注射时间点为运动后即刻,48 h组伊文氏蓝在运动后即刻和运动后24 h共注射2次。
(1)本研究N24组calpain3的mRNA表达显著高于C组(图3C),与Murphy等(2011)发现离心运动短期内激活calpain3的结果一致,说明大强度离心运动能够激活calpain3,但这不是本研究要证明和关注的内容,故并未展开讨论。
(2)是一个巨大蛋白,由3685个氨基酸组成,分子量约为427 kD。
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