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Altered phosphorylation status of ERK1/2 in soleus muscle of rat after tail suspension YuanMing , Jiang shi-zhong , Li zhi-li, Wang de-sheng ( Institute of space Medico-Engineering, Email:[email protected] ) Beijing 100094 Abstract: Objective To study the changes of ERK1/2 phosphorylation status induced by unloading of soleus muscle after tail suspension in rats. Method Hindlimb unweighting was produced by tail suspension, which can get simulated weightlessness effect, in male Wistar rats. Western blot was performed to detect the total and phospho-ERK1/2 status in isolated soleus muscle of 7d, 14d HU and respective control groups. Result Total ERK1/2 in soleus muscle was not affected by 7d and 14d-HU. The ERK1 phosphorylation status was declined significantly after 7d and 14d-HU.The ERK2 phosphorylation status was declined significantly after 7d-HU, but not for 14d-HU. Conclusion The unloading of soleus muscle after simulated microgravity can decrease the phosphorylation status of ERK1/2, which may influence the activation of transcription factors in the nucleus and contribute to the atrophy of soleus muscle induced by simulated weightlessness. Key words: weightlessness simulation; soleus muscle; Extracellualr signal-regulated kinase; mitogen-activated protein kinase 尾部悬吊模拟失重可改变比目鱼肌中 ERK1/2 的磷酸化状态. 袁明,姜世忠,李 志利,汪德生. 航天医学与医学工程 摘要:目的 观察尾吊模拟失重后大鼠比目鱼肌中 ERK1/2 磷酸化状态的变化。 方法 采用尾部悬吊大鼠模拟失重效应,以 western blot 技术检测游离的大鼠比目 鱼肌中总的 ERK1/2 含量和其磷酸化状态的变化。结果 7 天和 14 天尾吊模拟失 重后,大鼠比目鱼肌内总 ERK1/2 的含量没有发生改变。然而 ERK1 磷酸化状态 在 7 天和 14 天模拟失重后均显著降低。ERK2 的磷酸化状态在尾吊 7 天后显著 下降,但在 14 天尾吊后其下降程度却未达显著性。结论 尾吊模拟失重后可降低 大鼠比目鱼肌内 ERK1/2 的磷酸化状态,影响核内转录因子的激活,从而导致比 目鱼肌发生萎缩性变。 关键词:模拟失重;比目鱼肌;细胞外信号调节激酶;丝裂原活化蛋白激酶 中图分类号: 文献标识码: 文章编号: 作者地址:袁明. 航天医学工程研究所,北京 100094 Spaceflight and simulated weightlessness have been shown to cause atrophy, reduced functional capacity in limb muscles, with the greatest change observed in antigravity muscle such as the soleus muscle.[1,2] The atrophy primarily results from a reduced protein synthesis that is possibly initiated by the removal of the antigravity load. During spaceflight or simulated weightlessness, the sustained reductions in muscle activity result in a rapid and dramatic reduction in muscle fiber size resulted from alterations in gene expression. Criswell et al[3] reported that soleus troponin Ⅰslow(TnIs) mRNA was significantly decreased by 7d-hindlimb unweighting (HU) in mice. Moreover, Babjj and Booth[4] reported a 60 and 29% decline in soleus α-actin mRNA expressed per muscle and per microgram RNA after 7 days of HU. But for myosin, Thomason et al[5] found no decrease in soleus β-MHC mRNA with 7 days of HU. The hypothesis was developed that myosin and actin content are regulated independently and that their synthesis rate during spaceflight and simulated weightlessness are controlled by different mechanisms. The recent findings in 17-day spaceflight (STS-78)[2] showed a disproportionate loss of actin filaments in human soleus muscle. Thick-filament density and spacing were unchanged, whereas thin-filament density decreased significantly in overlap A-band region. Molecular studies indicate that the reduced protein synthesis induced by unloading can be attributed to the downregulation of gene expression. However, how unloading altered gene expression, protein synthesis and induced atrophy of antigravity muscles is a question to be answered. In recent years, the research of machanotransduction has been developed rapidly. Machanotransduction is the fundamental mechanism by which mechanical stress acting through a cell initiates intracellular signaling. Through the mechanism, forces promote cellular growth and survival[6]. All adhesion-dependent cells appear to be sensitive to mechanical forces, such as vascular smooth cells, cardiomyocytes, and skeletal muscle cells. Striated muscles are particularly responsive to mechanical stress, as evidenced by the fact that cellular size is in large port dictated by physical force: loading elicits hypertrophy whereas unloading elicits atrophy. The mitogen-activated protein kinase (MAPK) cascades involve in mechanically induced signaling from the cytosol to the nucleus[7]. There they phosphorylate many transcription factors, regulating gene expression[8] and protein synthesis, resulting in hypertrophy of cells. The MAPK family can be divided into five subfamilies, including ERK1/2, p38 MAPK, Jun NH2-terminal kinase(JNK), ERK3/4 and ERK5. Among the five subfamilies, the biological function of ERK1/2, p38 MAPK and JNK are better known. Especially, ERK1/2 has direct effect on muscle cells, activating several myogenic transcription factors, such as p90 ribsosomals c kinase(p90RSK), MAPK-interacting kinase 1(MnK 1) and eukaryotic initiation factor 4E(eIF4E), regulating muscle proliferation and development.[9] The purpose of this investigation was to examine differences, under control conditions and unloading conditions after 7d and 14d HU, in the total amount and phosphorylation of ERK1/2 in soleus muscles of rats. We tested the hypothesis that unloading induced by 7d and 14d HU would result in decrease of phosphorylated ERK1/2 proteins, which may affect indirectly the subsequent signaling pathways in nucleus, contributing to the atrophy of antigravity muscles. Methods Animal care and suspension procedure Male Wistar rats weighting 200-250g were obtained from Beijing Laboratory Animal Research Center. These animals were separately caged in an air-conditioned room maintained at 23℃ with a 12:12-h light-dark cycle. After housed for a minimum of 5 days, animals were randomly assigned to 7d-control, 14d-control, 7d-HU and 14d-HU treatment groups, with 4 rats in each group. HU was achieved by use of a tail harness to suspend the hindlimbs above the floor of the cage according to the method modified by Chenjie et al [10] as follows. The tail was cleaned and dried. A base layer of adhesive tape was attached laterally along the proximal two-thirds of the tail. The tape was then secured by wrapping the taped portion with three tape strips. The triangle hook attached to a swivel which tied with monofilament line was secured to the base layer of adhesive tape. Monofilament line tied to the swivel, which can pass through the small hole in the pillar of the cage, was used to raise the animals’ hindlimbs off the grid floor of cage. The height of the hook was adjusted to prevent the animals’ hindlimbs from touching the grid floor or other supportive surfaces, tilting the body about 35º, allowing the forelimbs to maintain contact with the grid floor and the animal to move about in a circular area to gain access to food and water. The exposed tip of tail was monitored daily to ensure adequate blood flow. The animals of control group were housed in standard cages in the same room as HU animals. All control and HU groups were fed a similar ad libitum diet of laboratory chow and tap water. Tissue dissection and preparation Rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (40mg/kg body mass). The left soleus muscle was removed, trimmed of connective tissue and fat, then frozen in liquid nitrogen and stored at -70℃ until further preparation and analysis. The skeletal muscle was homogenized in ice-cold buffer[11](50mM HEPES, PH 7.4, 4mM EGTA,10mM EDTA, 15mM Na4P2O7, 100mM β-glycerophosphate, 25mM NaF, 5mM Na3VO4, 0.1% Trixon-100) containing cocktail of protease inhibitors and 2mM phenylmethylsulfonyl fluoride(PMSF). After homogenization, the sample was vortexed for 30s and placed on ice for 30 min. Throughout the 30-min period, the sample was frequently vortexed, followed by clarification ( Centrifugation at 14000g, 4℃ for 15min). The supernatant was recovered and stored at -70℃. Protein concentration was determined by a BCA protein assay kit (Pierce, USA) with bovine serum albumin as a standard in a 96-well plate and reader (Bioteck, USA). Electrophoresis and western blot The sample containing 50 μg of total protein was boiled for 5min in loading buffer(62.5 mM Tris, 10% glycerol, 2% SDS, 50mM DTT, 0.01% bromphenol blue ), then was loaded onto 10% SDS-PAGE gel and electrophoresed at a constant current of 40 mA on a mini electrophoresis apparatus (Hoefer SE 250). After electrophoresis, proteins were transferred to a nitrocellulose membrane (Hybond) at 4℃ using a blotting unit (CBS, USA). The membrane was then washed with TBS(20mM Tris-HCl, 137mM NaCl, PH 7.6) for 5min at room temperature, blocked with 5% milk in TBST(1×TBS containing 0.1% Tween-20) for 1h (gently agitation at room temperature) and probed with either phospho-specific antibody for ERK1/2(1:1000) or antibody for total ERK1/2(1:1000, Cell Signal Technology) overnight at 4℃. After washing 3 times for 5min each in TBST, the membrane was then incubated for 1h at room temperature with a horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:2000, Santa Cruz Biotechnology). The protein bands were detected by chemiluminescence with LumiGLO (Cell Signal Technology), exposing the membrane to Kodak X-omat K film. Films were quantified by densitometry using a scanner and Quantity one Image software (Bio-Rad). The phosphorylation status of ERK1/2 was reported as densitometry volume of phospho-ERK1/2 divided by the densitometry volume for the total ERK1/2. Statistical analysis Data were expressed as means±SE. Statistical evaluation was done by ANOVA. Post hoc analysis was performed by using Newman-Keuls post hoc analysis. Differences between groups were considered statistically significant when P<0.05. Result Effects of 7d and 14d-HU on total ERK1/2 After 7d and 14d-HU, the total ERK1 and ERK2 were not significantly different compared with those of 7d and 14d-Con, respectively.(Fig3 and Fig4) The results indicated that the proteins expression of total ERK1/2 were not affected by unloading of soleus muscle which was induced by 7d and 14d-HU. Effects of 7d and 14d-HU on phosphorylation status of ERK1/2 Compared with 7d-Con, unloading of soleus muscle induced by 7d-HU resulted in a dramatic decreased phosphorylation status of ERK1 and ERK2 (only 51.15% and 63.1% of control respectively, Fig5 and Fig6). The differences of ERK1/2 phosphorylation status between two groups were significant (P<0.01). After 14d-HU, ERK1 phosphorylation status in soleus muscle was significantly less than that of control (P<0.01). But for ERK2, the phosphorylation status was declined to 77.43% of control, whereas the significant difference was not reached (P>0.05). Moreover, when compared with the ERK1/2 phosphorylation status of 7d-HU, there had an increasing trend in that of 14d-HU. These results indicated that the unloading of soleus muscle induced by HU can change the phosphorylation status of ERK1/2, which can subsequently affect the downstream signaling regulating the activity of various transcription factors in the nucleus. Fig 1.Effects of 7d and 14d-HU on total ERK1/2 Fig 2. Effects of 7d and 14d-HU on phospho-ERK1/2 Fig3. Effects of 7d and 14d-HU on total ERK1 (n=4) Fig4. Effects of 7d and 14d-HU on total ERK2 (n=4) Fig5. Effects of 7d and 14d-HU on phospho-ERK1 **P<0.01, *P<0.05, as compared with 7d-Con and 14d-Con respectively (n=4) Fig6. Effects of 7d and 14d-HU on phospho-ERK2 **P<0.01, as compared with 7d-Con (n=4) Discussion During spaceflight, exposure to the microgravity environment induced atrophy of antigravity muscles associated with reduced functional capacity, which was mostly suffered by astronauts when they returned to the 1-G environment. Many studies were focused on gaining a clear understanding of microgravity-induced changes in structure and function of antigravity muscles. So far, many important progress understanding these changes have been made.[12,13,14] However, compared with the knowledge of structure and functional changes induced by microgravity, the understanding of cellular and molecular mechanisms responsible for muscle atrophy is too little. As we know, both microgravity and simulated weightlessness can unload or reduce the mechanical stimulation on antigravity muscles, inducing the wasting and atrophy of muscles. Although we do not understand the mechanism responsible for mechanical unloading induced atrophy of muscles, some evidences showed that MAPK cascades are involved in mechanically induced signaling from cytosol to nucleus in many types of cell[11,15,16], regulating muscle hypertrophy in response to mechanical stimulation. In this study, we detected the changes of ERK1/2 phosphorylation status in soleus muscle of rats experiencing 7d and 14d-HU handling, testing the hypothesis that ERK1/2 signaling pathway participates in regulating mechanism responsible for atrophy of antigravity muscles induced by mechanical unloading. The results showed that HU handling, especially for 7d-HU handling , can significantly decrease the ERK1/2 phosphorylation status, but not changing the total ERK1/2 protein expression. After 14d-HU, ERK1/2 phosphorylation status still significantly decreased, whereas compared with 7d-HU, it had an increasing trend. Maybe this reflects an underlying autoregulation of muscle cells to adapt the new environment. To the best of our knowledge the present results are the first to show the inhibitory effect of simulated weightlessness on ERK1/2 signaling in soleus muscle. We detected the Thr202/Tyr204 phosphorylation as an endogenous reporter of ERK1/2 activity. Dual phsophorylation of ERK1/2 results in a conformational change and activating its kinase activity. Subsequently, the phosphorylated ERK1/2 can be translocated to the nucleus, regulating the activity of various transcription factors, promoting protein synthesis and muscle hyperthophy. MAPK signaling pathways are well known for their roles in regulating gene transcription and protein synthesis, however, more and more evidences indicated that MAPK signaling pathways participate in mechanotransduction[17,18] in recent years. In skeletal muscle, MAPK signaling pathways are considered as key pathways regulating hypertrophy of muscle induced by overload and resistance exercise[11,9]. Therefore, it is important to elucidate MAPK signaling mechanisms responsible for atrophy of antigravity muscles induced by weightlessness, which may show new lights on developing effective exercise countermeasures to prevent the atrophy of antigravity muscles. [References] [1] Caiozzo VJ, Baker MJ, Herrick RE. Effect of spaceflight on skeletal muscle: mechanical properties and myosin isoform content of a slow muscle [J]. Journal of applied physiology,1994,76():1764-1773 [2] Widrick JJ, knuth ST, Norenberg KM. Effect of a 17 day spaceflight on contractile properties of human soleus muscle fibers [J]. Journal of physiologist (Lond), 1999, 516():915-930 [3] Oriswel DS, Hodgson VRM, Hardeman EC. Nerve-response troponin Ⅰslow promoter does not respond to unloading [J]. 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