PO-104 The shifted balance of high-energy phosphates drives the AMPK dephosphorylation and reduced slow myosin expression during unloading

Objective Mechanical unloading of postural muscles results in both muscle atrophy and a slow to fast fibre type transition. The cause of such changes is a reduction in slow-type MyHC isoform expression and an increase in fast-type MyHC isoform expression. It is believed that calcineurin/NFAT signalling pathway and AMP-activated protein kinase (AMPK) are involved in the regulation of slow-type MyHC isoform. Previously we showed that AMPK phosphorylation is significantly decreased in rat soleus at the early stage of mechanical unloading. We found, that stimulation of AMPK activity at the early stage of gravitational unloading prevents a decline of slow MyHC expression. We assumed that a decrease in AMPK activity in rat soleus at the early stage of gravitational unloading could be associated with changes in the ratio of  intracellular high-energy  phosphates (ATP/ADP). The aim of the study was to investigate the  impact of  high-energy phosphates ratio changes on  AMPK activity and  slow-type MyHC isoform expression in rat soleus muscle at the early stages of gravitational unloading. Methods To verify the hypothesis, we used administration of β-guanidinopropionic acid (βGPA), before (6 day) and during 24-h hindlimb suspension. The content of phospho-AMPK, phospho-ACC, phospho-PKD, HDAC4/5  in rat soleus was determined by Western-blotting. The amount of MyHCI(β) pre-mRNA and mRNA was evaluated by RT-PCR.  Results After 24-h HS we observed a decrease (p<0.05) in phospho-AMPK content vs. control group, but in HS+ βGPA group didn’t differ from the control. After 24-h unloading we found a significant  increase in the content of nuclear HDAC4 in the HS group, but in the HS+ βGPA group the content of nuclear HDAC4 didn’t differ from the control group.  24-h unloading resulted in a decrease in MyHCI(β) pre-mRNA and mRNA expression vs. the control group. The expression level of MyHCI(β) pre-mRNA and mRNA in HS+ βGPA group didn’t differ from the control. Conclusions Thus βGPA administration prevents a decline in AMPK phosphorylation. Therefore, we can conclude that at the early stage of gravitational unloading an accumulation of high-energy phosphates  (ATP, ADP and creatine phosphate) takes place and leads to reduced AMPK activity and a slow to fast myosin fibre type transition. The study was supported by Russian Science Foundation grant # 18-15-00107

PO-064 A potential role of stretch-activated channels in anabolic mechanotransduction in the atrophied rat soleus muscle

Objective Prolonged immobilization or unloading of skeletal muscle causes muscle disuse atrophy, which is characterized by a reduction in muscle cross-sectional area and compromised contractile function. To date, the mechanisms of anabolic mechanotransduction in the atrophied mammalian skeletal muscle remain poorly understood. The aim of the present study was to assess a possible role of stretch-activated ion channels (SAC) in the propagation of a mechanical signal to anabolic signaling and protein synthesis (PS) in an isolated rat soleus muscle following mechanical unloading. Methods The mechanical unloading was performed via hindlimb suspension (HS). Twenty-eight male Wistar rats weighing were randomly assigned to the following 4 groups (n=7/group): 1) vivarium control (C), 2) control + SAC inhibitor (gadolinium) (C+Gd3+), 3) 7-day HS (HS), 4) 7-day HS + SAC inhibitor (HS+Gd3+). Following unloading, an isolated rat soleus was placed in an organ culture medium and subjected to a bout of eccentric contractions (EC). Upon completion of the EC, muscles were collected for Western blot analyses to determine the content of the key anabolic markers. The rate of PS was measured by SUnSET technique. Results EC-induced increase in PS was significantly less in the HS and HS+ Gd3+ groups vs. the C group. There was no statistically significant difference between the HS and HS+ Gd3+ groups in terms of EC-induced increase in muscle PS. A decrease in EC-induced phosphorylation of p70S6K, 4E-BP1, RPS6 and GSK-3beta in the 7-day unloaded soleus treated with SAC inhibitor did not differ from that of the 7-day unloaded soleus without SAC blockade. Thus, the inhibition of SAC with gadolinium did not lead to further decline in EC-induced phosphorylation of the key anabolic markers and muscle PS. Conclusions The results of the study suggest that attenuation of mTORC1-signaling and PS in response to EC in unloaded soleus muscle may be associated with inactivation of SAC. The study was supported by the RFBR grant # 16-34-60055

PL - 032 Physiological triggers involved in reduced slow myosin expression in disused postural muscle

Objective It is well known, that the reduced contractile activity of the postural soleus muscle under bedrest, immobilization or a space flight leads to decrease of slow myosin heavy chain (MyHC) expression rate and increase of the fast myosin isoforms expression [Pette, 2003; Stevens, 1999, 2000 et al]. The significant decline of the slow myosin mRNA content was found as early as after 24 hours of rat hindlimb unloading [Giger et al., 2009]. However, in the meantime, the mechanisms of this process had been substantially unexplored. At the same time, the main pathways involved in the control of transcription of Myh7 gene (MyHCIβ) are well known. These mechanisms are based upon traffic of messenger molecules (NFATc1 and Class IIA histone deacetylases) transducing positive and negative signals for Myh7 gene expression in muscle fiber nuclei. This traffic is known to be triggered by myoplasmic calcium content. Almost nothing is known about the roles of other physiological regulators (nitric oxide and high-energy phosphates) in Myh7 transcription control [Martins et al., 2012; Putman et al., 2015]. Our study was aimed to disclose the physiological triggers involved in the decline of Myh7 expression in postural muscle at the early stages of disuse state. We supposed that at the early stage of unloading (24 hours) it was the shift of the ATP/ADP/AMP balance (ATP accumulation due to muscle inactivation) to drive the Myh7 gene expression decline via AMP-activated protein kinase (AMPK) dephosphorylation and HDAC4 myonuclear import. Then we supposed that the mechanisms involved in the reduction of Myh7 expression during the first week of disuse are implemented via the decrease of NO muscle content [Lomonosova et al., 2011] and subsequent NFATc1 nuclear export in the GSK3β-dependent manner [Lomonosova et al, 2017].   Methods Three experimental series were performed in order to testify the hypotheses. Unloading of the hindlimbs was induced by using a standard rodent hindlimb suspension/unloading (HU) model (Morey-Holton & Globus, 2002). During the first series, using the selective AMPK activator AICAR we evaluated the roles of the AMPK dephosphorylation during the first days of unloading which we found earlier [Vilchinskaya et al., 2015; Mirzoev et al, 2016]. Animals were daily treated with AICAR (400 mg kg−1) or saline for 6 days before HU as well as during 24 h of HU.  The second series was designed to investigate the impact of high-energy phosphates ratio changes on AMPK activity and  slow-type MyHC isoform expression in rat soleus muscle at the early stages of unloading.  It is known that administration of β-guanidin-propionic acid (βGPA) allows shifting ATP/ADP/AMP balance to the enhanced ATP breakdown. We used administration of β-guanidinopropionic acid (βGPA), before (6 day) and during 24-h HU. The third series was aimed to identify the functional relationship between the decrease of the nitric oxide (NO) content, the GSK-3β phosphorylation (leading to the GSK-3β activation), the NFATc1 amount in the muscle nuclei, and the MyHC I(β) isoform expression in the rat soleus muscle under gravitational unloading. Male Wistar rats were divided into five groups: the vivarium control group; the group of animals with a 7-day hind limb suspension receiving placebo; the group of HU animals receiving a NO donor (L-arginine); the group of HU animals receiving a NO donor and a NO-synthase inhi bitor (L-NAME) and the group of HU animals receiving a GSK-3β inhibitor. Results In the 1st experimental series we discovered that AICAR treatment prevented a decrease in content of phospho-AMPK and pre-mRNA and mRNA expression of MyHC I as well as MyHC IIa mRNA expression. Twenty-four hours of HU resulted in HDAC4 accumulation in the nuclei of rat soleus but AICAR pretreatment prevented this accumulation. The results of the study indicate that AMPK dephosphorylation after 24 h of HU had a significant impact on the MyHC I and MyHC IIa mRNA expression in rat soleus. AMPK dephosphorylation also contributed to HDAC4 translocation to the nuclei of soleus muscle fibers, suggesting an important role of HDAC4 as an epigenetic regulator in the process of myosin phenotype transformation. In the 2nd experimental series after 24-h HS we observed a decrease (p<0.05) in phospho-AMPK content vs. control group, but in HS+ βGPA group didn’t differ from the control. After 24-h unloading we found a significant  increase in the content of nuclear HDAC4 in the HS group, but in the HS+ βGPA group the content of nuclear HDAC4 didn’t differ from the control group.  24-h unloading resulted in a decrease in MyHCI(β) pre-mRNA and mRNA expression vs. the control group. The expression level of MyHCI(β) pre-mRNA and mRNA in HS+ βGPA group didn’t differ from the control. Thus, βGPA administration prevents a decline in AMPK phosphorylation. Therefore, we can conclude that at the early stage of gravitational unloading an accumulation of high-energy phosphates  (ATP, ADP and creatine phosphate) may lead to reduced AMPK activity and a slow to fast myosin fiber type transition. The third experimental series dealt with the fate of the nuclear NFATc1 transcription factor which triggers the myh7 expression but can be easily exported from myonuclei being phosphorylated. It is supposed that it is NO-dependent GSK3β that phosphorylates NFATc1 and promotes its nuclear export.  We have shown that a 7-day unloading leads to a NO content decrease in the soleus muscle, and this effect is prevented by L-arginine administration. In addition, administration of L-arginine blocks the GSK-3β phosphorylation decrease, NFATc1 export from the muscle nuclei, and MyHC I(β) expression decrease caused by unloading. The NO-synthase inhibitor can block the L-arginine effect in each case. Administration of the GSK-3β inhibitor prevents the unloading-induced NFATc1 export from the muscle nuclei and a decrease of the MyHC I(β) expression. Conclusions The data obtained in the described experimental series give evidence for the novel view on the well-known phenomenon of slow-to-fast fiber type transition during unloading/disuse. It is obvious that the signaling pathways involved in the slow myosin gene expression control during unloading are time-dependent and consecutive in the course of the exposure to unloading. The earliest triggering factor is supposedly the shift of the balance of high-energy mononucleotide phosphates leading to decrease of AMP and accumulation of ATP content. This signal is accepted by the AMPK as a universal energy sensor and transduced to the transcription level by the altered HDAC4 traffic. It seems possible that at the next stages of the exposure to unloading the alteration of the calcineurin/NFATc1 signaling pathway takes place due to the activated calcineurin inhibitors [Lomonosova et al, 2017] and enhanced NFATc1 phosphorylation and myonuclear export. We obtained the novel evidence that at these stages the decline of Myh7 expression might be provided by the GSK3β activation and NFATc1 phosphorylation due to the decrease of NO content in the soleus muscle.  The study was supported by Russian Science Foundation grant # 18-15-00107.&nbsp

PO-094 p38 MAPK controls of E3-ligases expression and soleus atrophy attenuation in rat upon hindlimb unloading

Objective Unloading causes rapid skeletal muscle atrophy mainly due to the increased protein degradation. Muscle proteolysis results from the activation of ubiquitin-proteasome systems. The ubiquitination proteins are carried out by muscle-specific E3 ubiquitin ligases – MuRF-1 and MAFbx. It is known that MuRF-1 and MAFbx expression significantly increases on the third day of muscle unloading. We tested the hypothesis that p38 MAPK participates in the regulation of E3 ligases expression and the development of skeletal muscle atrophy during unloading. To check this idea we inhibited p38 MAPK by VX-745. Methods 21 male Wistar rats were divided into 3 groups (7 rats in each group): intact control (C), rats suspended for 3 days (HS) and rats suspended and injected i.p. with VX-745 (10 mg/kg/day) (VX). The hindlimb suspension was carried out according to Morey-Holton technique. The animals were anaesthetised with an i.p. injection of tribromoethanol (240 mg/kg). Under anesthesia, the m.soleus were excised, frozen in liquid nitrogen, and stored at -80°C until further analysis. 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. The statistical analysis was performed using the REST 2009 v.2.0.12 and Origin Pro programs at the significance level set at 0,05. The results are given as median in percent and interquartile range (0.25-0.75). Results The muscle weight in HS group was significantly reduced (72,3±2,5 mg) compared to C (83,0±3 mg), p<0.05, while the soleus weight of VX group didn’t differ from the control (84.2±5 mg). The MuRF1 mRNA expression was elevated dramatically in HS group (165 (138-210) %) when compared with the control (100 (64.6-112.5) %), p<0.05.  In the VX group the level of MuRF1 mRNA expression (127 (105-138) %) didn’t differ from the control group. The MAFbx mRNA expression was observed to increase equally in both suspended groups (294 (265-342) % and (271 (239-309) %).) vs C (100 (91-106) %) so, VX-745 administration did not have any significant effect on its expression. We also found that the level of ubiquitin mRNA expression in the soleus of HS rats was higher (423 (325-485) %) in comparison with the C group (100 (78-166) %, p<0.05) while VX-745 injection prevented increasing the  mRNA ubiquitin expression (200 (190-237) %). We discovered that the elevation of calpain-1 mRNA expression upon HS was prevented by VX-745 administration and its level didn’t differ from the control group (C - 100 (97-105) %, HS – 120 (116-133) %, VX - 107 (100-115) %, p<0.05). Conclusions Thus, the results indicate that the p38 MAPK signaling pathway takes part in the regulation of E3-ligase MuRF1 but not MAFbx expression. The p38 MAPK inhibition prevents muscle atrophy and the elevation of ubiquitin and calpain mRNA expression at the early stage of hindlimb unloading. This work was supported by RFBR grant No.17-04-01838

PO-121 Influence of HDAC1 inhibitor on the E3-ligases expression in rat soleus during hindlimb unloading

Objective Muscle unloading leads to its atrophy development. The MuRF-1 and MAFbx E3-ligases expression is increasing under this condition. FOXO3 was considered to be the only transcription factor that triggers E3-ligases expression. Beharry A.W. et al pinpoints HDAC1 as a primary regulator of FoxO in skeletal muscle that is both sufficient and required for skeletal muscle atrophy. We aimed to determine the role of histone deacetylase 1 (HDAC1) proteins in activation of MuRF-1 and MAFbx E3-ligases expression at the early stage of muscle unloading. Methods We investigate it by CI-994 (inhibitor of HDAC1) administration in male Wistar rats (180-200 g) upon 3-day hindlimb suspension. The method of hindlimb suspension was described in Morey-Holton E & Globus R (2002). 24 animals were divided into 3 groups (n=8 in each): C-control, CI - hindlimb suspension with CI-994 (i.p. 1 mg/kg/day), or placebo (HS group) administration. The animals were anaesthetized with an i.p. injection of tribromoethanol (240 mg/kg), soleus muscles were surgically excised, frozen in liquid nitrogen. The Western blot and RT-PCR analyzes were done. The statistical analysis was performed using REST 2009 v.2.0.12 and Bio-Rad CFX Manager programs at the significance level set at 0.05. The significant differences between groups were statistically analyzed using Mann-Whitney test. Results The evaluation of the levels of mRNA expression of MuRF-1 and MAFbx showed that CI-994 treatment inhibited unloading induced up-regulation of MAFbx in CI group but had no effect on mRNA expression of MuRF-1. After unloading, mRNA expression of MAFbx increased 2.12-fold (p < 0.05) in HS group. There were statistically significant differences in MAFbx mRNA expression between HS and CI groups. When compared with the control, unloading increased MuRF-1 mRNA expression  1.67- and 1.56-fold in HS and CI groups, respectively. CI-994 treatment also inhibited unloading-induced upregulation of mRNA expression of ubiquitin. The levels of ubiquitin mRNA expression when compared with the control were 4.21- and 2.32-fold in HS and CI groups, respectively. We did not find any differences in the content of phosphorylated anabolic signaling system components (Akt/mTOR/S6k) between both suspended groups (CI and HS). Conclusions Therefore, HDAC1 inhibiting prevented hindlimb suspension-induced up-regulation of MAFbx and ubiquitin, but did not any effect MuRF-1expression. This work was supported by Russian Science Foundation (grant № 18-15-00062)

PO-095 Effects of dynamic foot stimulation on GSK-3 beta signaling pathway in rat soleus muscle under hindlimb unloading

Objective It is known that under simulated microgravity GSK-3 beta phosphorylation (Ser 9) is decreased [Mirzoev et al., 2016] which is associated with the activation of its kinase activity. GSK-3 beta activation may lead to NFATc1 export from myonuclei resulting in a slow-to-fast myosin shift in soleus muscle and may cause a decrease in muscle oxidative capacity and protein synthesis [Glass, 2003, Theeuwes et al, 2017].  It was demonstrated that GSK-3 beta Ser 9 phosphorylation is decreased in response to nitric oxide synthase inhibition [Martins et al., 2011]. Under unloading conditions, the content of NO as well as nitric oxide synthase is attenuated in rat soleus muscle [Tidball et al., 1998; Lomonosova et al, 2011]. Dynamic foot stimulation (DFS) of the soles of the feet results in an increase of neuromuscular activation [Muller et al., 2005] of the lower limb muscles and prevents nitric synthase content decrease during 7-day exposure to dry immersion [Moukhina et al, 2004; Shenkman et al., 2004]. The aim of our study was to analyze the effect of rat dynamic foot stimulation during early unloading on GSK-3 beta phosphorylation and some of its downstream targets. Methods Male Wistar rats were randomly assigned to vivarium control, 1-day unloading and 1-day unloading with DFS. The pressure stimulation protocol mimicked the normal animal walking (104 mm Hg pressure, 4 Hz frequency and 250 ms signal duration) for a total of 20 min followed by a 10-min rest interval for 4 hours. Results We found that 1-day unloading caused a significant decrease (p˂ 0.025) in NFATc1 nuclear content as well as slow myosin (MHC I (beta) isoform) mRNA expression. In the 1-day unloading group, the level of glycogen synthase 1 phosphorylation (Ser 641), which is a direct GSK-3 beta target, was significantly higher than that in the control group, although a decrease in GSK-3 beta Ser 9 phosphorylation in the 1-day unloaded group was not statistically significant compared to the control group. However, in the DFS-treated unloaded rats the level of GSK-3 beta phosphorylation was significantly higher than in the untreated unloaded rats. NFATc1 nuclear content, slow myosin mRNA expression and glycogen synthase 1 phosphorylation (Ser 641) did not differ from the control group. The glycogen content in soleus muscles of both unloaded groups was higher than in the control group, which is in accordance with previous studies [Henriksen and Tischler, 1988], but in DFS subjected unloaded group the glycogen content was higher compared to the untreated unloaded group. Conclusions   Thus, we found that dynamic foot stimulation during 1-day hindlimb unloading leads to GSK-3 beta inactivation and prevention of both NFATc1 myonuclei export and the decrease in MyHC I beta expression. The study was supported by the Russian Foundation for Basic Research grant #17-29-1029 and Basic Research Program of the IBMP, RAS. The authors express their deep gratitude to Prof. Inessa B. Kozlovskaya for useful recommendations concerning the design of the study

P38α-MAPK Signaling Inhibition Attenuates Soleus Atrophy during Early Stages of Muscle Unloading

To test the hypothesis that p38α-MAPK plays a critical role in the regulation of E3 ligase expression and skeletal muscle atrophy during unloading, we used VX-745, a selective p38α inhibitor. Three groups of rats were used: non-treated control (C), 3 days of unloading/hindlimb suspension (HS), and 3 days HS with VX-745 inhibitor (HSVX; 10 mg/kg/day). Total weight of soleus muscle in HS group was reduced compared to C (72.3 ± 2.5 vs 83.0 ± 3 mg, respectively), whereas muscle weight in the HSVX group was maintained (84.2 ± 5 mg). The expression of muscle RING-finger protein-1 (MuRF1) mRNA was significantly increased in the HS group (165%), but not in the HSVX group (127%), when compared with the C group. The expression of muscle-specific E3 ubiquitin ligases muscle atrophy F-box (MAFbx) mRNA was increased in both HS and HSVX groups (294% and 271%, respectively) when compared with C group. The expression of ubiquitin mRNA was significantly higher in the HS (423%) than in the C and HSVX (200%) groups. VX-745 treatment blocked unloading-induced upregulation of calpain-1 mRNA expression (HS: 120%; HSVX: 107%). These results indicate that p38α-MAPK signaling regulates MuRF1 but not MAFbx E3 ligase expression and inhibits skeletal muscle atrophy during early stages of unloading

Differences in the Role of HDACs 4 and 5 in the Modulation of Processes Regulating MAFbx and MuRF1 Expression during Muscle Unloading

Unloading leads to skeletal muscle atrophy via the upregulation of MuRF-1 and MAFbx E3-ligases expression. Reportedly, histone deacetylases (HDACs) 4 and 5 may regulate the expression of MuRF1 and MAFbx. To examine the HDAC-dependent mechanisms involved in the control of E3-ubiquitin ligases expression at the early stages of muscle unloading we used HDACs 4 and 5 inhibitor LMK-235 and HDAC 4 inhibitor Tasqinimod (Tq). Male Wistar rats were divided into four groups (eight rats per group): nontreated control (C), three days of unloading/hindlimb suspension (HS) and three days HS with HDACs inhibitor LMK-235 (HSLMK) or Tq (HSTq). Treatment with LMK-235 diminished unloading-induced of MAFbx, myogenin (MYOG), ubiquitin and calpain-1 mRNA expression (p < 0.05). Tq administration had no effect on the expression of E3-ligases. The mRNA expression of MuRF1 and MAFbx was significantly increased in both HS and HSTq groups (1.5 and 4.0 folds, respectively; p < 0.05) when compared with the C group. It is concluded that during three days of muscle unloading: (1) the HDACs 4 and 5 participate in the regulation of MAFbx expression as well as the expression of MYOG, ubiquitin and calpain-1; (2) the inhibition of HDAC 4 has no effect on MAFbx expression. Therefore, HDAC 5 is perhaps more important for the regulation of MAFbx expression than HDAC 4

Role of Pannexin 1 ATP-Permeable Channels in the Regulation of Signaling Pathways during Skeletal Muscle Unloading

Skeletal muscle unloading results in atrophy. We hypothesized that pannexin 1 ATP-permeable channel (PANX1) is involved in the response of muscle to unloading. We tested this hypothesis by blocking PANX1, which regulates efflux of ATP from the cytoplasm. Rats were divided into six groups (eight rats each): non-treated control for 1 and 3 days of the experiments (1C and 3C, respectively), 1 and 3 days of hindlimb suspension (HS) with placebo (1H and 3H, respectively), and 1 and 3 days of HS with PANX1 inhibitor probenecid (PRB; 1HP and 3HP, respectively). When compared with 3C group there was a significant increase in ATP in soleus muscle of 3H and 3HP groups (32 and 51%, respectively, p < 0.05). When compared with 3H group, 3HP group had: (1) lower mRNA expression of E3 ligases MuRF1 and MAFbx (by 50 and 38% respectively, p < 0.05) and MYOG (by 34%, p < 0.05); (2) higher phosphorylation of p70S6k and p90RSK (by 51 and 35% respectively, p < 0.05); (3) lower levels of phosphorylated eEF2 (by 157%, p < 0.05); (4) higher level of phosphorylated GSK3β (by 189%, p < 0.05). In conclusion, PANX1 ATP-permeable channels are involved in the regulation of muscle atrophic processes by modulating expression of E3 ligases, and protein translation and elongation processes during unloading

How Postural Muscle Senses Disuse? Early Signs and Signals

A mammalian soleus muscle along with other &ldquo;axial&rdquo; muscles ensures the stability of the body under the Earth&rsquo;s gravity. In rat experiments with hindlimb suspension, zero-gravity parabolic flights as well as in human dry immersion studies, a dramatic decrease in the electromyographic (EMG) activity of the soleus muscle has been repeatedly shown. Most of the motor units of the soleus muscle convert from a state of activity to a state of rest which is longer than under natural conditions. And the state of rest gradually converts to the state of disuse. This review addresses a number of metabolic events that characterize the earliest stage of the cessation of the soleus muscle contractile activity. One to three days of mechanical unloading are accompanied by energy-dependent dephosphorylation of AMPK, accumulation of the reactive oxygen species, as well as accumulation of resting myoplasmic calcium. In this transition period, a rapid rearrangement of the various signaling pathways occurs, which, primarily, results in a decrease in the rate of protein synthesis (primarily via inhibition of ribosomal biogenesis and activation of endogenous inhibitors of mRNA translation, such as GSK3&beta;) and an increase in proteolysis (via upregulation of muscle-specific E3-ubiquitin ligases)