异丙肾上腺素在C2C12细胞分化中的作用及机制
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摘要
该研究主要探讨异丙肾上腺素(isoprenaline,ISO)在C2C12细胞分化与肌萎缩中的作用及可能的机制。在利用免疫组化分析C2C12细胞肾上腺素能受体表达特征的基础上,以2%的马血清高糖培养基建立C2C12细胞分化的实验体系,随后分别按单次或连续单次给予10~(–5) mol/L ISO后观察其分化差异;接着再比较连续单次给予不同浓度的ISO(10~(–8) mol/L、10~(–7) mol/L、10~(–6) mol/L、10~(–5) mol/L)处理;利用细胞免疫荧光化学和Western blot方法检测C2C12细胞分化后肌球蛋白重链(myosin heavy chain,MYH)的水平,并定量分析分化后骨骼肌细胞的肌管细胞核融合数目;同时,利用Western blot检测C2C12细胞分化调节有关的肌细胞生成蛋白(myogenin,Myo G)、p-p38MAPK(phosphorylated p38-mitogen-activated protein kinase)及p-AKT(phosphorylated/protein kinase B)的水平变化。结果显示,C2C12细胞呈现出α-肾上腺素和β-肾上腺素能受体表达的特征。分化培养诱导下,C2C12细胞随着时间的延长,分化成肌管的数量逐渐增多,在第8 d达峰值。单次一次给予10~(–5) mol/L ISO没有连续单次给予ISO抑制C2C12细胞分化成骨骼肌细胞显著,且连续单次给予ISO随着其浓度的增加,C2C12细胞分化为肌管细胞核融合数目逐渐减少并在ISO浓度为10~(–5) mol/L时最显著。与此同时,随着连续单次给予不同浓度(10~(–8) mol/L、10~(–7) mol/L、10~(–6) mol/L、10~(–5) mol/L)的ISO,MYH和Myo G的表达水平呈现随着浓度增加而逐渐降低的特征,而且p-p38MAPK及p-AKT的水平也呈连续单次给予ISO浓度增加而降低的趋势,在ISO浓度为10~(–5) mol/L时降低最显著。该研究提示,连续单次给予ISO显著抑制C2C12细胞分化为成熟骨骼肌细胞,且与p-p38MAPK、p-AKT及Myo G水平的降低密切相关,从而为交感神经长期过度兴奋所伴随的肌萎缩提供新的研究模型和治疗靶点。
This study is to investigate the effect and possible mechanism of isoprenaline(ISO) on C2C12 cells differentiation into skeletal muscle cells and muscle atrophy. Adrenergic receptors expressions in C2C12 cells were detected using immunofluorescence cytochemistry staining, and the cultured C2C12 cells that reached 70% confluence in vitro were used to establish the model of skeletal muscle stem/progenitor cells differentiation under 2% horse serum with high glucose DMEM. And then, using the cells differentiation model to compare the difference of either single or continuous single aderministration, single-dose(only one time) or continuous single-dose(one time each day for 8 days) of ISO with 10~(–5) mol/L were added into the differentiation medium. Subsequently, the different dosages effects of ISO with 10~(–8) mol/L, 10~(–7) mol/L, 10~(–6) mol/L or 10~(–5) mol/L on the cells differentiation were performed by using aderministration of continuous single-dose. After that, immunofluorescence cytochemistry staining were used to analyze the expression of myosin heavy chain(MYH) in differentiated cells from C2C12 cells, and the quantitative analysis of the number of nucleus of myotubes of skeletal muscle cells. Western blot was used to detect the expression of p-p38MAPK(phosphorylated p38-mitogen-activated protein kinase), p-AKT(phosphorylated protein kinase B), MYH and myogenin(Myo G). C2C12 cells showed the traits of α1-, α2-, β1-and β2-subtypes of adrenergic receptors expressions. C2C12 cells gradually differentiated into mature skeletal muscle cells during the procedure of differentiation condition, reaching the peak at day 8 of differentiation period. Singledose ISO with 10~(–5) mol/L slightly inhibited C2C12 cells differentiations into myotubes compared to continuous single-dose ISO. Furthermore, C2C12 cells differentiations into myotubes were gradually inhibited when exposed to continuous single-dose ISO with 10~(–8) mol/L, 10~(–7) mol/L, 10~(–6) mol/L, 10~(–5) mol/L, showing dose-dependent manner. Furthermore, the numbers of myotubes with the different nuclear fusion numbers were gradually reduced when the differentiating C2C12 cells were exposed to the stimulation of different dosages of continuous singledose ISO. Especially at the 10~(–5) mol/L continuous single-dose ISO, the potential of differentiation of C2C12 cells into mature skeletal muscle cells were markedly weaken. Simultaneously, the levels of p-p38 MAPK and p-AKT, and the expressions of MYH and Myo G as determined by Western blot were dosages-dependently decreased in the differentiating C2C12 cells with aderministration of continuous single-dose ISO. It is suggested that ISO, especially with continuous aderministration, inhibited C2C12 cells differentiation into mature skeletal muscle cells, which was associated with the reduced p-p38 MAPK and p-AKT levels, and decreased Myo G expressions. These results provided a new therapeutic target for prolonged sympathetic overactivity-related muscle atrophy.
This study is to investigate the effect and possible mechanism of isoprenaline(ISO) on C2C12 cells differentiation into skeletal muscle cells and muscle atrophy. Adrenergic receptors expressions in C2C12 cells were detected using immunofluorescence cytochemistry staining, and the cultured C2C12 cells that reached 70% confluence in vitro were used to establish the model of skeletal muscle stem/progenitor cells differentiation under 2% horse serum with high glucose DMEM. And then, using the cells differentiation model to compare the difference of either single or continuous single aderministration, single-dose(only one time) or continuous single-dose(one time each day for 8 days) of ISO with 10~(–5) mol/L were added into the differentiation medium. Subsequently, the different dosages effects of ISO with 10~(–8) mol/L, 10~(–7) mol/L, 10~(–6) mol/L or 10~(–5) mol/L on the cells differentiation were performed by using aderministration of continuous single-dose. After that, immunofluorescence cytochemistry staining were used to analyze the expression of myosin heavy chain(MYH) in differentiated cells from C2C12 cells, and the quantitative analysis of the number of nucleus of myotubes of skeletal muscle cells. Western blot was used to detect the expression of p-p38MAPK(phosphorylated p38-mitogen-activated protein kinase), p-AKT(phosphorylated protein kinase B), MYH and myogenin(Myo G). C2C12 cells showed the traits of α1-, α2-, β1-and β2-subtypes of adrenergic receptors expressions. C2C12 cells gradually differentiated into mature skeletal muscle cells during the procedure of differentiation condition, reaching the peak at day 8 of differentiation period. Singledose ISO with 10~(–5) mol/L slightly inhibited C2C12 cells differentiations into myotubes compared to continuous single-dose ISO. Furthermore, C2C12 cells differentiations into myotubes were gradually inhibited when exposed to continuous single-dose ISO with 10~(–8) mol/L, 10~(–7) mol/L, 10~(–6) mol/L, 10~(–5) mol/L, showing dose-dependent manner. Furthermore, the numbers of myotubes with the different nuclear fusion numbers were gradually reduced when the differentiating C2C12 cells were exposed to the stimulation of different dosages of continuous singledose ISO. Especially at the 10~(–5) mol/L continuous single-dose ISO, the potential of differentiation of C2C12 cells into mature skeletal muscle cells were markedly weaken. Simultaneously, the levels of p-p38 MAPK and p-AKT, and the expressions of MYH and Myo G as determined by Western blot were dosages-dependently decreased in the differentiating C2C12 cells with aderministration of continuous single-dose ISO. It is suggested that ISO, especially with continuous aderministration, inhibited C2C12 cells differentiation into mature skeletal muscle cells, which was associated with the reduced p-p38 MAPK and p-AKT levels, and decreased Myo G expressions. These results provided a new therapeutic target for prolonged sympathetic overactivity-related muscle atrophy.
引文
1 Sun Z,Liu L,Liu N,Liu Y.Muscular response and adaptation to diabetes mellitus.Front Bio sci 2008;13:4765-94.
2 Li Y,Zhang S,Zhang X,Li J,Ai X,Zhang L,et al.Blunted cardiac beta-adrenergic response as an early indication of cardiac dysfunction in Duchenne muscular dystrophy.Cardiovasc Res2014;103(1):60-71.
3 Middlekauff HR.Making the case for skeletal myopathy as the major limitation of exercise capacity in heart failure.Circ Heart Fail 2010;3(4):537-46.
4 Brum PC,Bacurau AV,Cunha TF,Bechara LR,Moreira JB.Skeletal myopathy in heart failure:Effects of aerobic exercise training.Exp Physiol 2014;99(4):616-20.
5 Lymperopoulos A,Rengo G,Koch WJ.Adrenergic nervous system in heart failure:Pathophysiology and therapy.Circ Res2013;113(6):739-53.
6 Bacurau AV,Jardim MA,Ferreira JC,Bechara LR,Bueno CR Jr,Alba-Loureiro TC,et al.Sympathetic hyperactivity differentially affects skeletal muscle mass in developing heart failure:Role of exercise training.J Appl Physiol 2009;106(5):1631-40.
7 Voltarelli VA,Bechara LR,Bacurau AV,Mattos KC,Dourado PM,Bueno CR Jr,et al.Lack ofβ2-adrenoceptors aggravates heart failure-induced skeletal muscle myopathy in mice.J Cell Mol Med 2014;18(6):1087-97.
8 Davel AP,Ceravolo GS,Wenceslau CF,Carvalho MH,Brum PC,Rossoni LV.Increased vascular contractility and oxidative stress inβ2-adrenoceptor knockout mice:the role of NADPH oxidase.J Vasc Res 2012;49(4):342-52.
9 Bueno CR Jr,Ferreira JC,Pereira MG,Bacurau AV,Brum PC.Aerobic exercise training improves skeletal muscle function and Ca2+handling-related protein expression in sympathetic hyperactivity-induced heart failure.J Appl Physiol 2010;109(3):702-9.
10 Medeiros A,Rolim NP,Oliveira RS,Rosa KT,Mattos KC,Casarini DE,et al.Exercise training delays cardiac dysfunction and prevents calcium handling abnormalities in sympathetic hyperactivity-induced heart failure mice.J Appl Physiol 2008;104(1):103-9.
11 Florea VG,Cohn JN.The autonomic nervous system and heart failure.Circ Res 2014;114(11):1815-26.
12 Martinez PF,Okoshi K,Zornoff LA,Carvalho RF,Oliveira Junior SA,Lima AR,et al.Chronic heart failure-induced skeletal muscle atrophy,necrosis,and changes in myogenic regulatory factors.Med Sci Monit 2010;16(12):BR374-83.
13 Burniston JG,Tan LB,Goldspink DF.beta2-adrenergic receptor stimulation in vivo induces apoptosis in the rat heart and soleus muscle.J Appl Physiol 2005;98(4):1379-86.
14 Zanou N,Gailly P.Skeletal muscle hypertrophy and regeneration:Interplay between the myogenic regulatory factors(MRFs)and insulin-like growth factors(IGFs)pathways.Cell Mol Life Sci2013;70(21):4117-30.
15 Bertaglia RS,Reissler J,Lopes FS,Cavalcante WL,Carani FR,Padovani CR,et al.Differential morphofunctional characteristics and gene expression in fast and slow muscle of rats with monocrotaline-induced heart failure.J Mol Histol 2011;42(3):205-15.
16 Ciciliot S,Rossi AC,Dyar KA,Blaauw B,Schiaffino S.Muscle type and fiber type specificity in muscle wasting.Int J Biochem Cell Biol 2013;45(10):2191-9.
17 Bacurau AV,Jannig PR,de Moraes WM,Cunha TF,Medeiros A,Barberi L,et al.Akt/m TOR pathway contributes to skeletal muscle anti-atrophic effect of aerobic exercise training in heart failure mice.Int J Cardiol 2016;214:137-47.
18 Tran P,Ho SM,Kim BG,Vuong TA,Leem YE,Bae GU,et al.TGF-β-activated kinase 1(TAK1)and apoptosis signalregulating kinase 1(ASK1)interact with the promyogenic receptor Cdo to promote myogenic differentiation via activation of p38MAPK pathway.J Biol Chem 2012;287(15):11602-15.
19 Lee SJ,Hwang J,Jeong HJ,Yoo M,Go GY,Lee JR,et al.PKN2 and Cdo interact to activate AKT and promote myoblast differentiation.Cell Death Dis 2016;7(10):e2431.
20 Garcia-Guerra L,Vila-Bedmar R,Carrasco-Rando M,CrucesSande M,Martín M,Ruiz-Gómez A,et al.Skeletal muscle myogenesis is regulated by G protein-coupled receptor kinase2 .J Mol Cell Biol 2014;6(4):299-311.
2 Li Y,Zhang S,Zhang X,Li J,Ai X,Zhang L,et al.Blunted cardiac beta-adrenergic response as an early indication of cardiac dysfunction in Duchenne muscular dystrophy.Cardiovasc Res2014;103(1):60-71.
3 Middlekauff HR.Making the case for skeletal myopathy as the major limitation of exercise capacity in heart failure.Circ Heart Fail 2010;3(4):537-46.
4 Brum PC,Bacurau AV,Cunha TF,Bechara LR,Moreira JB.Skeletal myopathy in heart failure:Effects of aerobic exercise training.Exp Physiol 2014;99(4):616-20.
5 Lymperopoulos A,Rengo G,Koch WJ.Adrenergic nervous system in heart failure:Pathophysiology and therapy.Circ Res2013;113(6):739-53.
6 Bacurau AV,Jardim MA,Ferreira JC,Bechara LR,Bueno CR Jr,Alba-Loureiro TC,et al.Sympathetic hyperactivity differentially affects skeletal muscle mass in developing heart failure:Role of exercise training.J Appl Physiol 2009;106(5):1631-40.
7 Voltarelli VA,Bechara LR,Bacurau AV,Mattos KC,Dourado PM,Bueno CR Jr,et al.Lack ofβ2-adrenoceptors aggravates heart failure-induced skeletal muscle myopathy in mice.J Cell Mol Med 2014;18(6):1087-97.
8 Davel AP,Ceravolo GS,Wenceslau CF,Carvalho MH,Brum PC,Rossoni LV.Increased vascular contractility and oxidative stress inβ2-adrenoceptor knockout mice:the role of NADPH oxidase.J Vasc Res 2012;49(4):342-52.
9 Bueno CR Jr,Ferreira JC,Pereira MG,Bacurau AV,Brum PC.Aerobic exercise training improves skeletal muscle function and Ca2+handling-related protein expression in sympathetic hyperactivity-induced heart failure.J Appl Physiol 2010;109(3):702-9.
10 Medeiros A,Rolim NP,Oliveira RS,Rosa KT,Mattos KC,Casarini DE,et al.Exercise training delays cardiac dysfunction and prevents calcium handling abnormalities in sympathetic hyperactivity-induced heart failure mice.J Appl Physiol 2008;104(1):103-9.
11 Florea VG,Cohn JN.The autonomic nervous system and heart failure.Circ Res 2014;114(11):1815-26.
12 Martinez PF,Okoshi K,Zornoff LA,Carvalho RF,Oliveira Junior SA,Lima AR,et al.Chronic heart failure-induced skeletal muscle atrophy,necrosis,and changes in myogenic regulatory factors.Med Sci Monit 2010;16(12):BR374-83.
13 Burniston JG,Tan LB,Goldspink DF.beta2-adrenergic receptor stimulation in vivo induces apoptosis in the rat heart and soleus muscle.J Appl Physiol 2005;98(4):1379-86.
14 Zanou N,Gailly P.Skeletal muscle hypertrophy and regeneration:Interplay between the myogenic regulatory factors(MRFs)and insulin-like growth factors(IGFs)pathways.Cell Mol Life Sci2013;70(21):4117-30.
15 Bertaglia RS,Reissler J,Lopes FS,Cavalcante WL,Carani FR,Padovani CR,et al.Differential morphofunctional characteristics and gene expression in fast and slow muscle of rats with monocrotaline-induced heart failure.J Mol Histol 2011;42(3):205-15.
16 Ciciliot S,Rossi AC,Dyar KA,Blaauw B,Schiaffino S.Muscle type and fiber type specificity in muscle wasting.Int J Biochem Cell Biol 2013;45(10):2191-9.
17 Bacurau AV,Jannig PR,de Moraes WM,Cunha TF,Medeiros A,Barberi L,et al.Akt/m TOR pathway contributes to skeletal muscle anti-atrophic effect of aerobic exercise training in heart failure mice.Int J Cardiol 2016;214:137-47.
18 Tran P,Ho SM,Kim BG,Vuong TA,Leem YE,Bae GU,et al.TGF-β-activated kinase 1(TAK1)and apoptosis signalregulating kinase 1(ASK1)interact with the promyogenic receptor Cdo to promote myogenic differentiation via activation of p38MAPK pathway.J Biol Chem 2012;287(15):11602-15.
19 Lee SJ,Hwang J,Jeong HJ,Yoo M,Go GY,Lee JR,et al.PKN2 and Cdo interact to activate AKT and promote myoblast differentiation.Cell Death Dis 2016;7(10):e2431.
20 Garcia-Guerra L,Vila-Bedmar R,Carrasco-Rando M,CrucesSande M,Martín M,Ruiz-Gómez A,et al.Skeletal muscle myogenesis is regulated by G protein-coupled receptor kinase2 .J Mol Cell Biol 2014;6(4):299-311.