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


MAP-Kinase p38 Hindlimb unloading NFATc1


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 slow- type 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 slow- tonic 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 muscles. 1. Introduction 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 [22]. The muscle fiber type, in turn, determines the fiber charac- teristics, such as maximal contraction force, maximal contraction ve- locity and fatigue resistance [22]. Muscle unloading or inactivity under conditions of rat hindlimb unloading (suspension) [25] or human bed- rest [10] 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, 13,24]. Calcineurin/NFATc1 signaling pathway is one of the most well- known 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 T- lymphocytes, cytoplasmic 1) [14,26]. The calcium-activated phospha- tase calcineurin dephosphorylates NFATс1 and induces its nuclear translocation [6]. Inside the nuclei NFATc1 binds to MyHC I(β) pro- moter 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) [2] and p38 MAPK (p38) [5] 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 [11] and interact with MEF-2 transcriptional factors [16]. * Corresponding author. E-mail addresses: [email protected], [email protected] (K.A. Sharlo), [email protected] (E.P. Mochalova), [email protected] (S.P. Belova), [email protected] (I.D. Lvova), [email protected] (T.L. Nemirovskaya), [email protected] (B.S. Shenkman). https://doi.org/10.1016/j.abb.2020.108622 Received 3 June 2020; Received in revised form 16 September 2020; Accepted 5 October 2020 Available online 11 October 2020 0003-9861/© 2020 Elsevier Inc. All rights reserved. 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 [23]; 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 [7]. 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 sus- pension (3HS) and 3-day hindlimb suspension with daily oral supple- mentation of 10 mg/kg p38 MAPK inhibitor VX-745 (3HS VX-745). The hindlimb suspension was performed by Morey-Holton method [19]. 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, respec- tively), 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 Scien- tific, USA) according to manufacturers protocol with modifications. Complete Protease Inhibitor Cocktail (Santa-Cruz), Phosphatase Inhib- itor Cocktail B (Santa Cruz), PMSF (1 mM), aprotinin (10 lg/ml), leu- peptin (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, Gene- Tex, Inc., USA, # GTX59567), total p38 (1:500, Cell Signaling Tech- nology, USA, #9212), MEF-2D (1:1,000 AMD Millipore), TEAD1 (Cell signaling, 1:1,000), NFATc1 (Abcam, 1:1,000). All the primary anti- bodies were used for overnight incubation at 4 ◦C. The secondary HRP- conjugated antibodies (goat-anti-rabbit, Santa Cruz, 1: 30,000, goat- anti-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 pro- teins were detected were stripped with RestoreWestern Blot Stripping Buffer (Thermo Scientific) and then re-probed with total protein anti- bodies overnight at 4 ◦C to analyze the phosphorylation level of the proteins. Then the blots were incubated with HRP-conjugated goat-anti- rabbit secondary antibody and visualized as described above. It was controlled that phosphorylated proteins—goat-anti-rabbit-HRP com- plexes 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. 2.4. Immunohistochemisty The transverse frozen sections of the m. Soleus samples were pre- pared 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 fluo- rescent 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 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 tem- perature. PCR primers used for RNA analysis are as follows: Myh7 (MyHCI(β)) F: 5′-ACAGAGGAAGACAGGAAGAACCTAC-3’; R: 5′- GGGCTTCACAGGCATCCTTAG-3’; Myh2 (MyHCIIA) F: 5′-TATCCT- CAGGCTTCAAGATTTG-3’; R: 5′-TAAATAGAATCACATGGGGACA-3’; Myh4 (MyHCIIB) F: 5′-CTGAGGAACAATCCAACGTC-3’; R: 5′- 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 TTGTGTGATTTCTTCTGTCACCT-3’; Myh1(MyHCIId/X) F: 5′- (p < 0.05); & - significant differences from hindlimb suspended group. CGCGAGGTTCACACCAAA-3’; R: 5′-TCCCAAAGTCGTAAGTA- CAAAATGG-3’; Myh7b F: 5′-GAGTGTGGAGCAGGTGGTATTT-3’; R: 5′- GGACCCCAATGAAGAACTGA-3’; Rcan1 (MCIP1.4) F: 5′- CCGTTGGCTGGAAACAAG-3’; R: 5′-GGTCACTCTCACACACGTGG-3’; cyclophilin A F: 5′- AGCACTGGGGAGAAAGGATT-3’; R: 5′- CAATGC- CAACTCTCGTCAACAG-3’. The amplification was realtime monitored using SYBR Green I dye and the iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad Lab- oratories, 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 [12]. 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 dif- ferences 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. 3. Results 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 hindlimb- suspended animals partially prevented the p38 phosphorylation in- crease, 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 signifi- cantly 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 down- regulation 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 signifi- cantly 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 cho- sen to identify the level of NFATc1-dependent transcription, as the alternative promoter before the exon 4 of MCIP1 contains 15 NFAT- binding 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 phosphory- lation 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 (Fig. 5B). 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 [11], 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 con- tents 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. 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 % of control groups (mean ± SEM). * significant differences from control group (p < 0.05); & - significant differences from hindlimb suspended group. 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 [15]. myh7b mRNA does not translate into a protein molecule, but produces micro-RNA 499 [3]. 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 [15]. Basing on these facts we decided to analyze the contents of myh7b and SOX6 mRNAs in soleus muscles of the experimental animals. 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). 4. Discussion 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 [7]. 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 [29]. However, this suggestion still needs to be verified. 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 hin- dlimb suspension without affecting GSK-3β activity. The myosins mRNAs transcription pattern in the 3HS group corre- sponds 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. More- over, 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. How- ever, our data indicate that p38 MAP-kinase inhibition does not signif- icantly 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 NFAT- dependent MCIP1.4 mRNA transcription decline in hindlimb- suspended animals suggests that p38 downregulates slow MyHC tran- scription 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. 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. 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 statisti- cally significant (Fig. 3) [23]. It has been previously shown that NFATc1 nuclear content decreases dramatically after the first day and transiently increases after the third day of unloading [23]. Having regard to the above, the observed discrepancies between NFATc1 nuclear accumula- tion and MCIP1.4 mRNA transcription may be explained by a time-lag between NFAT nuclear translocation and target genes mRNA accumu- lation. 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 hindlimb unloading. 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 [28]. 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 group. 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. 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). 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 suspended group. 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 con- ducted and with the approved legal acts of the Russian Federation and international organizations. Declaration of competing interest The authors declare no conflict of interests. Acknowledgement 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 content). References [1] D.L. Allen, J.J. Uyenishi, A.S. Cleary, R.S. Mehan, S.F. Lindsay, J.M. Reed, Calcineurin activates interleukin-6 transcription in mouse skeletal muscle in vivo and in C2C12 myotubes in vitro, Am. J. Physiol. Regul. Integr. Comp. Physiol. 298 (2010) R198–R210. [2] C.R. Beals, C.M. Sheridan, C.W. Turck, P. Gardner, G.R. Crabtree, Nuclear export of NF-ATc enhanced by glycogen synthase kinase-3, Science 275 (1997) 1930–1934. [3] M.L. Bell, M. Buvoli, L.A. Leinwand, Uncoupling of expression of an intronic microRNA and its myosin host gene by exon skipping, Mol. Cell Biol. 30 (2010) 1937–1945. [5] J.C. Braz, O.F. Bueno, Q. Liang, B.J. Wilkins, Y.S. Dai, S. Parsons, J. Braunwart, B. J. Glascock, R. Klevitsky, T.F. Kimball, T.E. Hewett, J.D. Molkentin, Targeted inhibition of p38 MAPK promotes hypertrophic cardiomyopathy through upregulation of calcineurin-NFAT signaling, J. Clin. Invest. 111 (2003) 1475–1486. [6] E.R. Chin, E.N. Olson, J.A. Richardson, Q. Yang, C. Humphries, J.M. Shelton, H. Wu, W. Zhu, R. Bassel-Duby, R.S. Williams, A calcineurin-dependent transcriptional pathway controls skeletal muscle fiber type, Genes Dev. 12 (1998) 2499–2509. [7] F. Derbre, B. Ferrando, M.C. Gomez-Cabrera, F. Sanchis-Gomar, V.E. Martinez- Bello, G. Olaso-Gonzalez, A. Diaz, A. Gratas-Delamarche, M. Cerda, J. Vina, Inhibition of Xanthine oXidase by allopurinol prevents skeletal muscle atrophy: role of p38 MAPKinase and E3 ubiquitin ligases, PloS One 7 (2012), e46668. [8] D. Desplanches, M.H. Mayet, E.I. Ilyina-Kakueva, J. Frutoso, R. Flandrois, Structural and metabolic properties of rat muscle exposed to weightlessness aboard Cosmos 1887, Eur. J. Appl. Physiol. Occup. Physiol. 63 (1991) 288–292. [9] C.P. Ingalls, J.C. Wenke, R.B. Armstrong, Time course changes in [Ca2 ]i, force, and protein content in hindlimb-suspended mouse soleus muscles, Aviat Space Environ. Med. 72 (2001) 471–476. [10] B.S.S.I.B. Kozlovskaya, Cellular responses of human postural muscle to dry immersion, Front. Physiol. (2019). [11] K.C. Lin, T. Moroishi, Z. Meng, H.S. Jeong, S.W. Plouffe, Y. Sekido, J. Han, H. W. Park, K.L. Guan, Regulation of Hippo pathway transcription factor TEAD by p38 MAPK-induced cytoplasmic translocation, Nat. Cell Biol. 19 (2017) 996–1002. [12] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real- time quantitative PCR and the 2(-Delta Delta C(T)) Method, Methods 25 (2001) 402–408. [13] T.P. Martin, V.R. Edgerton, R.E. Grindeland, Influence of spaceflight on rat skeletal muscle, J. Appl. Physiol. 65 (1985) 2318–2325, 1988. [14] K.J. Martins, M. St-Louis, G.K. Murdoch, I.M. MacLean, P. McDonald, W.T. DiXon, C.T. Putman, R.N. Michel, Nitric oXide synthase inhibition prevents activity- induced calcineurin-NFATc1 signalling and fast-to-slow skeletal muscle fibre type conversions, J Physiol 590 (2012) 1427–1442. [15] J.J. McCarthy, K.A. Esser, C.A. Peterson, E.E. Dupont-Versteegden, Evidence of MyomiR network regulation of beta-myosin heavy chain gene expression during skeletal muscle atrophy, Physiol. Genom. 39 (2009) 219–226. [16] S.L. McGee, M. Hargreaves, EXercise and myocyte enhancer factor 2 regulation in human skeletal muscle, Diabetes 53 (2004) 1208–1214. [17] J.D. Meissner, P.K. Umeda, K.C. Chang, G. Gros, R.J. Scheibe, Activation of the beta myosin heavy chain promoter by MEF-2D, MyoD, p300, and the calcineurin/ NFATc1 pathway, J. Cell. Physiol. 211 (2007) 138–148. [18] T. Mirzoev, S. Tyganov, N. Vilchinskaya, Y. Lomonosova, B. Shenkman, Key markers of mTORC1-dependent and mTORC1-independent signaling pathways regulating protein synthesis in rat soleus muscle during early stages of hindlimb unloading, Cell. Physiol. Biochem. 39 (2016) 1011–1020. [19] E.R. Morey-Holton, R.K. Globus, Hindlimb unloading rodent model: technical aspects, J. Appl. Physiol. 92 (1985) 1367–1377, 2002. [20] A.M. Mukhina, E.G. Altaeva, T.L. Nemirovskaya, B.S. Shenkman, The role of L-type calcium channels in the accumulation of Ca2 in soleus muscle fibers in the rat and changes in the ratio of myosin and serca isoforms in conditions of gravitational unloading, Neurosci. Behav. Physiol. 38 (2008) 181–188. [21] B. Rothermel, R.B. Vega, J. Yang, H. Wu, R. Bassel-Duby, R.S. Williams, A protein encoded within the Down syndrome critical region is enriched in striated muscles and inhibits calcineurin signaling, J. Biol. Chem. 275 (2000) 8719–8725. [22] S. Schiaffino, C. Reggiani, Fiber types in mammalian skeletal muscles, Physiol. Rev. 91 (2011) 1447–1531. [23] K. Sharlo, I. Paramonova, O. Turtikova, S. Tyganov, B. Shenkman, Plantar mechanical stimulation prevents calcineurin-NFATc1 inactivation and slow-to-fast fiber type shift in rat soleus muscle under hindlimb unloading, J. Appl. Physiol. 126 (1985) 1769–1781, 2019. [24] L. Stevens, C. Firinga, B. Gohlsch, B. Bastide, Y. Mounier, D. Pette, Effects of unweighting and clenbuterol on myosin light and heavy chains in fast and slow muscles of rat, Am. J. Physiol. Cell Physiol. 279 (2000) C1558–C1563. [25] L. Stevens, B. Gohlsch, Y. Mounier, D. Pette, Changes in myosin heavy chain mRNA and protein isoforms in single fibers of unloaded rat soleus muscle, FEBS Lett. 463 (1999) 15–18. [26] J. Tothova, B. Blaauw, G. Pallafacchina, R. Rudolf, C. Argentini, C. Reggiani, S. Schiaffino, NFATc1 nucleocytoplasmic shuttling is controlled by nerve activity in skeletal muscle, J. Cell Sci. 119 (2006) 1604–1611. [27] R.W. Tsika, C. Schramm, G. Simmer, D.P. Fitzsimons, R.L. Moss, J. Ji, Overexpression of TEAD-1 in transgenic mouse striated muscles produces a slower skeletal muscle contractile phenotype, J. Biol. Chem. 283 (2008) 36154–36167. [28] M. Yamakuchi, I. Higuchi, S. Masuda, Y. Ohira, T. Kubo, Y. Kato, I. Maruyama, I. Kitajima, Type I muscle atrophy caused by microgravity-induced decrease of myocyte enhancer factor 2C (MEF2C) protein expression, FEBS Lett. 477 (2000) 135–140. [29] K. Yuasa, K. Okubo, M. Yoda, K. Otsu, Y. Ishii, M. Nakamura, Y. Itoh, K. Horiuchi, Targeted ablation of p38alpha MAPK suppresses denervation-induced muscle atrophy, Sci. Rep. 8 (2018) 9037.