The role of MAP-kinase p38 in the m. soleus slow myosin mRNA
transcription regulation during short-term functional unloading
K.A. Sharlo *
, E.P. Mochalova, S.P. Belova, I.D. Lvova, T.L. Nemirovskaya, B.S. Shenkman
Institute of Biomedical Problems, RAS, Moscow, 76A Khoroshevskoe Shosse, 123007, Russia
The unloading of postural muscles leads to the changes in myosins heavy chains isoforms (MyHCs) mRNAs
transcription pattern, that cause severe alterations of muscle functioning. Several transcription factors such as
NFATc1 and TEAD1 upregulate slow MyHC mRNA transcription, and p38 MAP kinase can phosphorylate NFAT
and TEAD1, causing their inactivation. However, the role p38 MAP kinase plays in MyHCs mRNAs transcription
regulation in postural soleus muscle during unloading remains unclear. We aimed to investigate whether
pharmacological inhibition of p38 MAPK during rat soleus unloading would prevent the unloading-induced slowtype MyHC mRNA transcription decrease by affecting calcineurin/NFATc1 or TEAD1 signaling. Male Wistar rats
were randomly assigned to three groups: cage control (C), 3-day hindlimb suspended group (3HS) and 3-day
hindlimb suspended group with the daily oral supplementation of 10 mg/kg p38 MAPK inhibitor VX-745
(3HS + VX-745). 3 days of hindlimb suspension caused the significant decreases of slow MyHC and slowtonic myh7b mRNAs transcription as well as the decrease of NFATc1-dependent MCIP1.4 mRNA transcription
in rat soleus muscles compared to the cage control. P38 MAP-kinase inhibition during hindlimb suspension
completely prevented slow MyHC mRNA content decrease and partially prevented slow-tonic myh7b and
MCIP1.4 mRNAs transcription decreases compared to the 3HS group. We also observed NFATc1 and TEAD1
myonuclear contents increases in the 3HS + VX-745 group compared to both 3HS and C groups (p < 0.05).
Therefore, we found that p38 inhibition counteracts the unloading-induced slow MyHC mRNA transcription
downregulation and leads to the activation of calcineurin/NFAT signaling cascade in unloaded rat soleus
The type of a skeletal muscle fiber is determined by the relative
contents of slow and fast myosin heavy chain (MyHC) isoforms in the
fiber . The muscle fiber type, in turn, determines the fiber characteristics, such as maximal contraction force, maximal contraction velocity and fatigue resistance . Muscle unloading or inactivity under
conditions of rat hindlimb unloading (suspension)  or human bedrest  as well as during space flight [8,13] result in the increases of
fast MyHCs mRNA content and the decrease of slow type I MyHC mRNA
content in soleus muscles. These changes lead to slow-to-fast shift and
cause severe alterations of the muscles metabolism and functioning [8,
Calcineurin/NFATc1 signaling pathway is one of the most wellknown pathways of type I MyHC mRNA transcription regulation. The
transcription of the type I MyHC mRNA (MyHC I(β)) in skeletal muscle
was shown to be activated by NFATc1 (nuclear factor of activated Tlymphocytes, cytoplasmic 1) [14,26]. The calcium-activated phosphatase calcineurin dephosphorylates NFATс1 and induces its nuclear
translocation . Inside the nuclei NFATc1 binds to MyHC I(β) promoter and activates MyHC I(β) mRNA transcription, cooperating with
other activators and coactivators of MyHC I(β) transcription, such as
MEF-2D or TEAD1 [17,27]. However, NFATc1 can be removed out of the
muscle nuclei by several protein kinases that phosphorylate NFATc1,
counteracting the activity of calcineurin. Glycogen synthase kinase 3β
(GSK-3β)  and p38 MAPK (p38)  both can phosphorylate NFATc1,
causing its exit from the nuclei in skeletal or cardiac muscles. P38 MAPK
can also phosphorylate MyHC I(β) transcription activator TEAD1 and
block its activity  and interact with MEF-2 transcriptional factors
* Corresponding author.
E-mail addresses: [email protected], [email protected] (K.A. Sharlo), mochalova_ekat[email protected] (E.P. Mochalova), [email protected] (S.P. Belova),
[email protected] (I.D. Lvova), nemirovska[email protected] (T.L. Nemirovskaya), [email protected] (B.S. Shenkman).
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics
journal homepage: www.elsevier.com/locate/yabbi
Received 3 June 2020; Received in revised form 16 September 2020; Accepted 5 October 2020
Archives of Biochemistry and Biophysics 695 (2020) 108622
The role of the unloading-induced GSK-3β activation in NFATc1
nuclear export and MyHC I(β) mRNA transcription regulation has been
described in previous studies ; however, the role of MAP-kinase p38
in MyHC I(β) transcription regulation and NFATc1 nucleo-cytoplasmic
shuttling during hindlimb unloading remains unclear, although p38 is
activated during unloading . Therefore, basing on these data we
aimed to investigate whether pharmacological inhibition of p38
MAP-kinase during hindlimb unloading would prevent the
unloading-induced decrease of slow MyHC mRNA transcription by
counteracting NFATc1 and TEAD1 nuclear export and inactivation.
2. Material and methods
2.1. Animal studies
All procedures with the animals were approved by the Biomedicine
Ethics Committee of the Institute of Biomedical Problems of the Russian
Academy of Sciences/Physiology section of the Russian Bioethics
Committee (protocol N◦ 500, January 23, 2019). All experiments were
performed in strict accordance with ARRIVE guidelines. Male Wistar
rats were randomly assigned to cage control (C), 3-days hindlimb suspension (3HS) and 3-day hindlimb suspension with daily oral supplementation of 10 mg/kg p38 MAPK inhibitor VX-745 (3HS + VX-745).
The hindlimb suspension was performed by Morey-Holton method .
Briefly, a strip of adhesive tape was applied to the animal’s tail, which
was suspended by passing the tape through a swivel that was attached to
a metal bar on the top of the cage. This allowed the forelimbs to have
contact with the grid floor and allowed the animals to move around the
cage for free access to food and water. The suspension height was
adjusted to prevent the hindlimbs from touching any supporting surface
while maintaining a suspension angle of approximately 30◦. After the
experiment, the rats were sacrificed by tribromethanol overdose, and
their soleus muscles were dissected and immediately frozen in liquid
nitrogen. Three days of unloading had no statistically significant effect
on the total body weight of the experimental rats. The average total body
weights were 200 ± 13.5, 186 ± 9.97, and 195 ± 12.9 g for C, 3HS, and
3HS + VX rats, respectively. Total weight of soleus muscle in the 3HS
group was reduced compared to C (72.3 ± 2.5 vs 83.0 ± 3 mg, respectively), whereas muscle weight in the 3HS + VX group was maintained
(84.2 ± 5 mg).
2.2. Nuclear and cytoplasmic extracts preparation
Nuclear extracts were prepared from 50 mg of soleus muscle using
NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, USA) according to manufacturers protocol with modifications.
Complete Protease Inhibitor Cocktail (Santa-Cruz), Phosphatase Inhibitor Cocktail B (Santa Cruz), PMSF (1 mM), aprotinin (10 lg/ml), leupeptin (10 lg/ml), and pepstatin A (10 lg/ml) were used to maintain
extract integrity and function. Nuclear extracts were dialyzed by means
of Amicon Ultra-0.5 centrifuge filters (Millipore, USA). The protein
content of all samples was quantified twice using a Quick Start Bradford
Protein Assay (Bio-Rad Laboratories) in order to calculate the optimal
sample value for electrophoretic gel. The supernatant fluid was diluted
with 2X sample buffer (5.4 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol,
10% β-mercaptoethanol, 0.02% bromphenol blue) and stored at − 85 ◦C
for immunoblot procedures. The quality of nuclear and cytoplasm
fractions separation was measured by immunoblot of GAPDH in nuclear
fraction – no visible bands were detected.
2.3. SDS-PAGE and immunoblots
To determine the levels NFATc1, MEF-2D and TEAD1 in rat soleus
nuclear extract and phosphorylated and total MAPK p 38, GSK-3β and
glycogen synthase 1 in cytoplasmic extract sodium dodecyl sulfate
(SDS)–polyacrylamide gel electrophoresis followed by Western blotting.
Electrophoresis was carried out in the 10% separating polyacrylamide
gel (0.2% methylene-bisacrylamide, 0.1% SDS, 375 mM Tris-HCl, pH
8.8, 0.05% ammonium persulfate, 0.1% TEMED) and in the 5%
concentrating polyacrylamide gel (0.2% methylene-bisacrylamide,
0.1% SDS, 125 mM Tris-HCl, pH 6.8, 0.05% ammonium persulfate,
0.1% TEMED). The cathode (192 mM Tris-glycine, pH 8.6, 0.1% SDS)
and anode (25 mM Tris-HCl, pH 8.6) buffers were used. Samples were
loaded at the rate of 25 μg of total protein in each sample. The samples of
each group were loaded on the gel together with control samples.
Electrophoresis was carried out at 17 mA/gel in a mini system (Bio-Rad
Laboratories) at room temperature.
Electrotransfer of the proteins was carried out in buffer (25 mM Tris,
pH 8.3, 192 mM glycine, 20% ethanol, 0.04% SDS) onto nitrocellulose
membrane at 100 V and 4 ◦C in the mini Trans-Blot system (Bio-Rad) for
120 min. The membranes were blocked in 5% non-fat dry milk solution
(Bio-Rad) in PBST (phosphate-buffered saline pH 7.4, 0.1% Tween 20)
for 1 h at room temperature. After the SDS-PAGE electrotransfer of the
proteins was carried out onto nitrocellulose membrane at 100 V and 4 ◦C
in the mini Trans-Blot system (Bio-Rad) for 120 min. To reveal protein
bands, the following primary polyclonal antibodies were used: total
GSK-3β and phosphorylated Ser 9 GSK-3β (Cell signaling, 1:1,000),
GAPDH (Cell Signaling, 1: 10,000), total glycogen synthase 1 and Ser
641 phosphorylated glycogen synthase 1 (Abcam, 1:10,000), lamin B1
(Abcam, 1:1,000), Tyr 180/Thr182 phosphorylated p38 (1:500, GeneTex, Inc., USA, # GTX59567), total p38 (1:500, Cell Signaling Technology, USA, #9212), MEF-2D (1:1,000 AMD Millipore), TEAD1 (Cell
signaling, 1:1,000), NFATc1 (Abcam, 1:1,000). All the primary antibodies were used for overnight incubation at 4 ◦C. The secondary HRPconjugated antibodies (goat-anti-rabbit, Santa Cruz, 1: 30,000, goatanti-mouse, Santa Cruz, 1: 25,000) were used for a 1-h incubation at
room temperature. The blots were revealed using the ImmunStar TM
Substrate Kit (Bio-Rad Laboratories, USA) and the C-DiGit Blot Scanner
(LI-COR Biotechnology, USA). The blots on which phosphorylated proteins were detected were stripped with RestoreWestern Blot Stripping
Buffer (Thermo Scientific) and then re-probed with total protein antibodies overnight at 4 ◦C to analyze the phosphorylation level of the
proteins. Then the blots were incubated with HRP-conjugated goat-antirabbit secondary antibody and visualized as described above. It was
controlled that phosphorylated proteins—goat-anti-rabbit-HRP complexes were washed out completely from the blots. The blots were
washed 3 × 10 min at room temperature with PBST after incubations
with antibodies and Restore Western Blot Stripping Buffer. The signals of
all protein bands from total protein fraction except for phosphorylated
proteins were normalized to GAPDH; phosphorylated proteins were
normalized to total proteins content. Nuclear fraction proteins signals
were normalized to lamin B1.
The transverse frozen sections of the m. Soleus samples were prepared by Leica CM 1900 cryostat (10 μm thick; Braunschweig, Germany)
at 20 ◦C, dried at room temperature for 15 min, and incubated in PBST
for 20 min. Sections were incubated with primary antibodies against
MyHCs fast—1:60, DSMZ or with primary antibodies against MyHC slow
(1:100, Sigma, St. Louis, MO) for 1 h at 37 ◦C. Anti-MyHCs fast antibody
used in this study does not distinguish between different fast MyHC
isoforms. The fibers that did not express fast MyHCs were accounted as
slow-type fibers, and the fibers that did not express slow-type MyHC
were accounted as fast-type fibers. After three 10-min washes with
PBST, the sections were incubated with secondary antibodies (Alexa
Fluor 350, 1: 1,000; Molecular Probes, Waltham, MA) for 60 min in the
dark at room temperature. After that, the sections were washed 3 times
with PBST, examined, and photographed with a Leica Q500MC fluorescent microscope at magnification × 200. The percentage of different
muscle fiber types was evaluated relative to all muscle fibers present in
each section. The sum of all myofibers detected exceeds 100% because
K.A. Sharlo et al.
Archives of Biochemistry and Biophysics 695 (2020) 108622
of double-positive hybrid fibers. At least 10 cross-sections per sample
were analyzed to determine the percentage of different muscle fiber
types in the sample.
2.5. RNA isolation and RT-qPCR
Total RNA was extracted from frozen soleus muscle samples using
RNeasy Micro Kit (Qiagen, Germany) according to the manufacturer’s
protocol. RNA concentration was analyzed at 260 nm. RNA quality of
purification was evaluated according to 260/280 and 260/230 ratios.
Reverse transcription was performed by incubation of 0.5 μg of RNA,
random hexamers d(N)6, dNTPs, RNase inhibitor and MMLV reverse
transcriptase for 60 min at 42 ◦C. MyHC I(β), MyHC II A, MyHC II B,
MyHC IId/x, MCIP1.4 and myh7b expression levels were determined by
real-time PCR. The samples to be compared were run under similar
conditions (template amounts, duration of PCR cycles). The annealing
temperature was based on the PCR primers’ optimal annealing temperature. PCR primers used for RNA analysis are as follows: Myh7
The amplification was realtime monitored using SYBR Green I dye
and the iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad Laboratories, USA). To confirm the amplification specificity, the PCR
products from each primer pair were subjected to a melting curve
analysis, and sequencing of the products was provided at least once.
Relative quantification was performed based on the threshold cycle (CT
value) for each of the PCR samples . peptidylprolyl isomerase A
(cyclophilin A) gene was tested and chosen for the normalization of all
quantitative PCR analysis experiments in the current study.
2.6. Statistical analysis
All values are shown as means ± SEM of 8 animals. The means of all
groups are shown as % of the control group. To check whether the differences among groups are statistically significant, we adopted the
Kruskal-Wallis nonparametric test, followed by Dunn’s post hoc test. A p
value less than 0.05 was regarded as statistically significant.
The level of MAP-kinase p38 Tyr 180/Thr182 phosphorylation
significantly increased after three days of hindlimb suspension in the
3HS group compared to control. VX-745 treatment of hindlimbsuspended animals partially prevented the p38 phosphorylation increase, so that in the 3HS + VX-745 group the phosphorylation of p38
was significantly lower compared to the 3HS group and did not differ
from C group (Fig. 1.).
After the three days of hindlimb suspension the slow-type MyHC I(β)
mRNA and the fast oxidative MyHC II A mRNA contents were significantly decreased, while fast glycolytic MyHC II B and MyHC IId/x
mRNAs contents were significantly increased in the 3HS group
compared to control group (p < 0.05), (Fig. 2A–D). At the same time
MyHC I(β) mRNA transcription in the 3HS + VX-745 group did not differ
from that of control and it was significantly higher compared to the 3HS
group (Fig. 2 A). The fast MyHC II A, MyHC II B and MyHC IId/x mRNAs
were not affected by inhibition of p38 and their contents did not differ
between the two hindlimb-suspended groups (Fig. 2B–D). Therefore,
MAP kinase p38 inhibition prevented the unloading-induced downregulation of the slow-type myosin I (β) mRNA transcription.
The analysis of the slow-to-fast fiber-type ratio showed that after
three days of hindlimb suspension the percentage of slow-type fibers
significantly decreased, and the percentage of fast-type fibers significantly increased in both 3HS and 3HS + VX-745 groups versus control
group (Fig. 3 b).
At the next stage of our work we analyzed the content of NFATc1 in
nuclear fraction of soleus muscles and the level of NFATc1-dependent
transcription activity. MCIP1.4 isoform mRNA transcription was chosen to identify the level of NFATc1-dependent transcription, as the
alternative promoter before the exon 4 of MCIP1 contains 15 NFATbinding sites [1,21].
The NFATc1 nuclear content slightly decreased after the three days
of hindlimb suspension (this decrease was not statistically significant),
while the transcription of MCIP1.4 mRNA dramatically decreased (by
82%) compared to cage control (p < 0.05). P38 inhibition led to the
accumulation of NFATc1 in soleus muscles myonuclear fraction, and
NFATc1 myonuclear content in the 3HS + VX-745 group significantly
excessed both cage control and 3HS groups levels (Fig. 4 A). However,
p38 inhibition only partially prevented MCIP1.4 mRNA transcription
decrease, although the differences between 3HS and 3HS + VX-745
groups were significant (Fig. 4B).
NFATc1 nuclear content as well as NFATc1-dependent transcription
may be regulated by GSK-3β, so we analyzed Ser 9 GSK-3β phosphorylation and glycogen synthase Ser 641 phosphorylation, which is the
direct downstream target of GSK-3β. The Ser 9 phosphorylation of GSK-
3β significantly decreased in both 3HS and 3HS + VX-745 groups
compared to control, while there were no differences between 3HS and
3HS + VX-745 groups (Fig. 5 A). At the same time, the level of glycogen
synthase 1 Ser 641 phosphorylation significantly increased in both
hindlimb-suspended groups compared to control, indicating the equal
level of GSK-3 β kinase activation in 3HS and 3HS + VX-745 groups
P38 MAP-kinase was shown to interact with TEAD1 and MEF-2D
transcription factors [16,27]. Both of these factors may regulate the
slow-type myosin mRNA transcription , so we analyzed the effect of
hindlimb suspension and p38 MAP-kinase inhibition on TEAD1 and
MEF-2D myonuclear contents. Both TEAD1 and MEF-2D nuclear contents were slightly lower in 3HS group compared to control group. In the
3HS + VX-745 group the TEAD1 nuclear content was significantly
higher compared to the 3HS and C groups. However, the MEF-2D
Fig. 1. MAP-kinase p38 Thr180/Tyr 182 phosphorylation. All data are shown
as % of control groups (mean ± SEM). C - cage control group, 3HS -hindlimb
suspended for 3 days group, 3HS + VX-745- hindlimb suspended for 3 days VX-
745 administered group (10 mg/kg) * significant differences from control group
(p < 0.05); & - significant differences from hindlimb suspended group.
K.A. Sharlo et al.
Archives of Biochemistry and Biophysics 695 (2020) 108622
nuclear content in the 3HS + VX-745 group was even lower than in the
3HS group and significantly differed from control group (Fig. 4. C, D).
MyHC I(β) mRNA in skeletal muscle fibers induces the transcription
of slow-tonic myosin myh7b gene by micro-RNA-dependent mechanisms
. myh7b mRNA does not translate into a protein molecule, but
produces micro-RNA 499 . This micro-RNA targets the 3′ UTR of the
transcriptional repressor SOX6, which is involved in the repression of
slow fiber type genes, in particular, MyHC I(β), and downregulates SOX6
mRNA transcription . Basing on these facts we decided to analyze
the contents of myh7b and SOX6 mRNAs in soleus muscles of the
myh7b mRNA content in the 3HS group was significantly decreased
compared to both control and 3HS + VX-745 groups (Fig. 6 A), while in
the 3HS + VX-745 group myh7b mRNA content was equal to control
(Fig. 6A). SOX6 mRNA content in the 3HS group was significantly
increased compared to control, and p38 inhibition partially prevented
SOX6 mRNA increase, so that in the 3HS + VX-745 group it did not
significantly differ from neither control nor 3HS group (Fig. 6B).
The observed p38 MAP-kinase phosphorylation increase after the
three days of rat hindlimb suspension corresponds to data by Derbre et
all, although in that work the changes were detected after two weeks of
hindlimb suspension . Since in the 3HS + VX-745 group the level of
MAP-kinase p38 phosphorylation was downregulated (Fig. 1) we
conclude that VX-745 administration in our experiment reached the goal
and led to the inhibition of unloading-induced MAP-kinase p38 activity.
Nevertheless, the mechanisms of p38 MAP-kinase activation during
unloading remain unknown. We suggest that the unloading-induced
increase of calcium ions in the sarcoplasm of the postural muscles,
which was observed in many studies as early as after the second day of
rat hindlimb suspension [9,20], may lead to the activation of p38 by
CaMKII-dependent phosphorylation and/or by myokine-dependent
MAP-kinase activation . However, this suggestion still needs to be
The observed GSK-3β Ser 9 phosphorylation decrease as well as
glycogen synthase 1 Ser 641 phosphorylation increase after 3 days of
hindlimb suspension in both 3HS and 3HS + VX-745 groups suggest that
the pharmacological inhibition of p38 did not influence GSK-3β activity,
so we can conclude that p-38 inhibition in our experiment contributed to
NFATc1 nuclear export and NFAT-dependent transcription during hindlimb suspension without affecting GSK-3β activity.
The myosins mRNAs transcription pattern in the 3HS group corresponds to literature data concerning myosins mRNA transcription under
conditions of the early stage of hindlimb unloading [15,18]. However,
the slow-tonic myh7b mRNA transcription downregulation and SOX6
mRNA increase as early as after three days of rat hindlimb suspension
were observed for the first time. P38 inhibition during three days of rat
hindlimb suspension prevented both slow MyHC and slow-tonic myh7b
mRNAs transcription decreases, so we can conclude that p38 activation
contributes to the unloading-induced slow myosins transcription
downregulation at the early stages of rat hindlimb suspension. Moreover, the downregulation of SOX6 mRNA transcription in the 3HS +
VX-745 group indicates that the unloading-induced p38 activation
contributes to SOX6 repressor transcription increase, so p38 may
contribute to the SOX6-dependent repression of slow-type genes. However, our data indicate that p38 MAP-kinase inhibition does not significantly prevent slow-to-fast fiber-type percentage decrease after three
days of rat hindlimb unloading. It could be possible that the effects of
p38 inhibition on slow-to-fast fiber-type ratio would manifest at later
stages of unloading.
The observation that p38 inhibition partially prevents of NFATdependent MCIP1.4 mRNA transcription decline in hindlimbsuspended animals suggests that p38 downregulates slow MyHC transcription by targeting calcineurin/NFATc1 signaling cascade. The effect
of p38 inhibition on nuclear accumulation of NFATc1 after three days of
hindlimb unloading indicates that p38 contributes to the nuclear export
Fig. 2. The myosins heavy chain isoforms mRNAs transcription. (a) MyHC I(β) mRNA; (b) MyHC IIA mRNA; (с) MyHC IIB mRNA, (d) MyHC IId/x mRNA. C - cage
control group, 3HS -hindlimb suspended for 3 days group, 3HS + VX-745- hindlimb suspended for 3 days VX-745 administered group, All data are shown as %
control groups (mean ± SEM). * significant differences from control group (p < 0.05); & - significant differences from hindlimb suspended group.
K.A. Sharlo et al.
Archives of Biochemistry and Biophysics 695 (2020) 108622
of NFATc1 under conditions of hindlimb unloading. However, we
cannot say that p38 inhibition prevented the unloading-induced
NFATc1 nuclear content decrease in soleus muscles after 3 days of rat
hindlimb suspension, as the decrease was far less profound than the
decline of MCIP1.4 mRNA transcription and actually was not statistically significant (Fig. 3) . It has been previously shown that NFATc1
nuclear content decreases dramatically after the first day and transiently
increases after the third day of unloading . Having regard to the
above, the observed discrepancies between NFATc1 nuclear accumulation and MCIP1.4 mRNA transcription may be explained by a time-lag
between NFAT nuclear translocation and target genes mRNA accumulation. So it may be possible that the observed protective effect of p38
inhibition on MCIP1.4 and slow MyHC mRNAs transcription is the result
of the NFATc1 nuclear content decrease prevention at 1–2 days of rat
The observed MEF-2D nuclear content decline is in accordance with
the previous data concerning the decline of MEF-2 nuclear content in
skeletal muscles after 14-days rat space flight . Nevertheless, the
MEF-2D nuclear export seems not to contribute much to the slow myosin
mRNA transcription in our experiment, at least in p38 inhibitor group.
However, the accumulation of TEAD1 in soleus muscle nuclear fraction,
which was observed in p38-inhibited hindlimb-suspended group, could
contribute to slow MyHC mRNA transcription upregulation in this
Therefore, it is concluded that MAP-kinase p38 contributes to the
unloading-induced slow MyHC transcription decrease in rat soleus
muscle after three days of unloading. P38 inhibition during unloading
counteracts both the downregulation of calcineurin/NFATc1 signaling
pathway and the increase of the transcriptional repressor of slow-type
genes SOX6 mRNA, possibly by myh7b-dependent mechanism, and
leads to accumulation of TEAD1 in soleus myonuclei. Thus, p38 may
also take part in the downregulation of calcineurin/NFATc1 signaling
pathway and the increase of SOX6 during unloading.
Fig. 3. The immunohistochemical analysis of the slow-to-fast fiber-type ratio. A – the microphotographs of anti-fast and anti-slow MyHCs immunostained CSAs. B –
slow-to-fast fiber-type ratio; C - cage control group, 3HS -hindlimb suspended for 3 days group, 3HS + VX-745- hindlimb suspended for 3 days VX-745 administered
group, All data are shown as % of control groups (mean ± SEM). There were analyzed 1762 fast MyHCs-negative (slow) fibers in C group, 1693 in 3HS group and
1527 in 3HS + VX-745 group. And there were analyzed 409 slow MyHC I negative (fast) fibers in C group, 462 in 3HS group and 618 in 3HS + VX-745 group*
significant differences from control group (p < 0.05); & - significant differences from hindlimb suspended group.
K.A. Sharlo et al.
Archivs of Biochemistry and Biophysics 695 (2020) 108622
Fig. 4. Transcription factors nuclear contents and MCIP1.4 mRNA transcription. C - cage control group, 3HS -hindlimb suspended for 3 days group, 3HS + VX-745-
hindlimb suspended for 3 days VX-745 administered group. (a) NFATc1 nuclear content; (b) MCIP1.4 mRNA; (c) TEAD1 nuclear content; (d) MEF-2D nuclear
content. * significant differences from control group (p < 0.05); & - significant differences from hindlimb suspended group.
Fig. 5. a – GSK-3β Ser 9 phosphorylation, b - glycogen synthase 1 Ser 641 phosphorylation. All data are shown as % of control groups (mean ± SEM). C - cage control
group, 3HS -hindlimb suspended for 3 days group, 3HS + VX-745- hindlimb suspended for 3 days VX-745 administered group. * significant differences from control
group (p < 0.05).
K.A. Sharlo et al.
Archives of Biochemistry and Biophysics 695 (2020) 108622
Compliance with ethical standards
All procedures performed in studies on animals were in compliance
with ethical standards of the institution in which the studies were conducted and with the approved legal acts of the Russian Federation and
Declaration of competing interest
The authors declare no conflict of interests.
Supported by RFBR N◦ 17-04-01838 А (experiment conduction and
protein and mRNA extraction) and Russian Science Foundation Grant
18-15-00107 (the analysis of gene expression and myonuclear proteins
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Fig. 6. myh7b and SOX6 mRNAs transcription. C - cage control group, 3HS -hindlimb suspended for 3 days group, 3HS + VX-745- hindlimb suspended for 3 days VX-
745 administered group, (a) myh7b mRNA; (b) SOX6 mRNA.* significant differences from control group (p < 0.05); & – significant differences from hindlimb
K.A. Sharlo et al.