Download Spaceflight and simulated microgravity have been shown to cause

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]. Journal of applied physiology, 1998,
84():1083-1087
[4] Babji P and Booth FW. α-actin and cytochrome c mRNA in atrophied adult rat
skeletal muscle [J]. American Journal of physiology cell physiology, 1988,
254():c651-c656
[5] Thomason DB, Brggs RB and Booth FW. Protein metabolism and β-myosin
heavy-chain mRNA in unweighted soleus muscle [J]. American Journal of
physiology Regulatory Integrative Comp Physiology, 1989,257():R300-R305
[6] Martineau LC and Phillip FG. Insight into skeletal muscle mechanotransduction:
MAPK activation is quantitatively related to tension [J]. Journal of applied
physiology, 2001,91():693-702
[7] Ikeda M, Takei T and Mills I. Calcium-independent activated of Extrecellular
signal-regulated kinase 1 and 2 by cyclic strain[J]. Biochem Biophys Res
Commun,1998,247():462-465
[8] Garrington TP and Johnson GL. Organization and regulation of mitogen-activated
protein kinase signaling pathways [J]. Curr Opin Cell Biol, 1999,11():211-218
[9] Williamson D, Gallagher P, Harber M. Mitogen-activated protein kinase (MAPK)
pathway activation: effects of age and acute exercise on human skeletal muscle [J].
Journal of physiologist, 2003, 547():977-987
[10] Chen Jie, Ma Jin, Ding Zhaoping, et al. A modified tail-suspension model for
simulating long-term weightlessness[J]. Chinese Journal of Space Science. 1993,
13(2):159-162
陈杰，马进，丁兆平 一种模拟失重影响的大鼠尾部悬吊模型[J]. 空间科学
学报，1993，13（2）：159－162
[11] Carlson, Christian J, Zhiqiang Fan, et al. Time course of the MAPK and
PI3-kinase response within 24h of skeletal muscle overload [J]. Journal of
applied physiology, 2001, 91():2079-2087
[12] Stelzer JE and Jeffrey JW. Effects of hindlimb suspension on the functional
properties of slow and fast soleus fibers from three strains of mice [J]. Journal of
applied phyisiology, 2003, 95():2425-2433
[13] Harrison BC, Allen DL, Girten B, et al. Skeletal muscle adaptations to
microgravity exposure in the mouse [J]. Journal of applied physiology, 2003,
95():2462-2470
[14] Tang Bin, Fan Xiao-li, Wu su-di. Effects of tail suspension on
electrophysiological characteristics of muscle spindles in isolated rat soleus
muscle [J]. Space medicine & Medical Engineering, 2004, 17(1):1-6
[15] Hung CT, Henshaw DR, Wang CC, et al. Mitogen-activated protein kinase
signaling in bovine articular chondrocytes in response to fluid flow does not
require calcium mobilization [J]. J.Biomech, 2000, 33():73-80
[16] Yamazaki T, Komuro I, Yazaki Y. Molecular aspects of mechanical
stress-induced cardiac hypertrophy [J]. Mol. Cell. Biochem, 1996,
164():197-201
[17] Daniel JT, Guohao Dai, Ivan VM, et al. Mechanotransduction through
growth-factor shedding into the extracellular space [J]. Nature, 2004,
429(5):83-86
[18] Krook A, Ulrika W, Xin Jian jiang , et al. Effects of exercise on mitogen and
stress-activated kinase signal transduction in human skeletal muscle [J].
Am.J.Physiol Regulatory Integrative Comp Physiol , 2000, 279():R1716-R1721