Natalia A. Vilchinskaya

It is well established that the early stage of mechanical unloading in both humans and animals is characterized by a decline in the activity of the key cellular energy sensor, AMP-activated protein kinase (AMPK). This decline triggers signaling pathways that drive myosin phenotype remodeling and muscle atrophy. However, the molecular mechanisms underlying the reduction in AMPK activity under these conditions remain poorly understood. We hypothesized that elevated activity of protein kinase D1 (PKD1) could contribute to the decline in AMPK activity during the initial stage of rat hindlimb unloading. The present study demonstrates that the suppression of upregulated PKD1 activity via a specific inhibitor (CRT0066101) during 24-hour unloading results in the restoration of AMPK activity in the rat soleus muscle. Thus, we have shown for the first time the contribution of PKD1 to the regulation of AMPK activity in the mammalian postural muscle during the initial stage of hindlimb unloading.

The main findings of our study are as follows: 1) The soleus slow fibers atrophy after 21-day dry immersion (DI) does not exceed that after 7-day DI; 2) The soleus ubiquitin ligases expression after 21-day DI returns to its initial level; 3) The soleus slow fibers atrophy after 21-day DI is accompanied by a mitochondrial apparatus structural markers decrease; 4) The soleus fibers signaling pathways restructuring process during 21-day DI is carried out in a complex manner.

Dry immersion dysregulates mitochondrial genes expression, affects mi-RNA expression and PGC1 promoter methylation.

Currently, no ideal treatment exists to combat skeletal muscle disuse-induced atrophy and loss of strength. Because the activity of AMP-activated protein kinase (AMPK) in rat soleus muscle is suppressed at the early stages of disuse, we hypothesized that pre-treatment of rats with metformin (an AMPK activator) would exert beneficial effects on skeletal muscle during disuse. Muscle disuse was performed via hindlimb suspension (HS). Wistar rats were divided into four groups: (1) control (C), (2) control + metformin for 10 days (C+Met), (3) HS for 7 days (HS), (4) metformin treatment for 7 days before HS and during the first 3 days of 1-week HS (HS+Met). Anabolic and catabolic markers were assessed using WB and RT-PCR. Treatment with metformin partly prevented an HS-induced decrease in rat soleus weight and size of slow-twitch fibers. Metformin prevented HS-related slow-to-fast fiber transformation. Absolute soleus muscle force in the HS+Met group was increased vs. the HS group. GSK-3β (Ser9) phosphorylation was significantly increased in the HS+Met group vs. the HS group. Metformin pre-treatment partly prevented HS-induced decrease in 18S+28S rRNA content and attenuated upregulation of calpain-1 and ubiquitin. Thus, pre-treatment of rats with metformin can ameliorate disuse-induced reductions in soleus muscle weight, the diameter of slow-type fibers, and absolute muscle strength.

The Maintenance of AMPK Activity Eliminates Abnormally Accelerated Differentiation of Primary Myoblasts Isolated from Atrophied Rat Soleus Muscle Поддержание нормальной активности AMPK устраняет аномально ускоренную дифференцировку первичных миобластов, выделенных из атрофированной m. soleus крысы

Regrowth of atrophied myofibers depends on muscle satellite cells (SCs) that exist outside the plasma membrane. Muscle atrophy appears to result in reduced number of SCs due to apoptosis. Given reduced AMP-activated protein kinase (AMPK) activity during differentiation of primary myoblasts derived from atrophic muscle, we hypothesized that there may be a potential link between AMPK and susceptibility of differentiating myoblasts to apoptosis. The aim of this study was to estimate the effect of AMPK activation (via AICAR treatment) on apoptosis in differentiating myoblasts derived from atrophied rat soleus muscle. Thirty rats were randomly assigned to the following two groups: control (C, n = 10) and 7-day hindlimb suspension (HS, n = 20). Myoblasts derived from the soleus muscles of HS rats were divided into two parts: AICAR-treated cells and non-treated cells. Apoptotic processes were evaluated by using TUNEL assay, RT-PCR and WB. In differentiating myoblasts derived from the atrophied soleus, there was a significant decrease (p < 0.05) in AMPK and ACC phosphorylation in parallel with increased number of apoptotic nuclei and a significant upregulation of pro-apoptotic markers (caspase-3, -9, BAX, p53) compared to the cells derived from control muscles. AICAR treatment of atrophic muscle-derived myoblasts during differentiation prevented reductions in AMPK and ACC phosphorylation as well as maintained the number of apoptotic nuclei and the expression of pro-apoptotic markers at the control levels. Thus, the maintenance of AMPK activity can suppress enhanced apoptosis in differentiating myoblasts derived from atrophied rat soleus muscle.

Mechanical unloading of skeletal muscles leads to the development of atrophic processes and a decrease in the total number of myosatellite cells involved in muscle regeneration. In vitro studies revealed an accelerated differentiation of myoblasts derived from the rat m. soleus after an unloading-induced decrease in AMP-activated protein kinase (AMPK). AMPK is required for the activation of SCs and also participates in the regulation of myoblast proliferation and differentiation. It can be hypothesized that a decrease in AMPK activity after mechanical unloading promotes the acceleration of myoblast differentiation. The main purpose of this study was to elucidate a possible role of AMPK in the regulation of differentiation of myoblasts isolated from the rat m. soleus after mechanical unloading. To test this hypothesis, a specific AMPK activator, AICAR, was used to prevent a decrease in AMPK activity during differentiation of myoblasts isolated from the rat m. soleus after 7-day unloading. Immunocytochemistry, PCR-RT and Western blotting were used to assess changes during myoblast differentiation. In differentiating myoblasts derived from the unloaded m. soleus, there was a significant decrease in AMPK (Thr172) and ACC (Ser79) phosphorylation levels, an increase in myotube differentiation index, as well as in the expression of myoblast fusion factors and myogenic regulatory factors (MRF). Furthermore, there was a decrease in the expression of slow myosin heavy chains (MyHC) and an increase in the expression of fast MyHC isoforms. AICAR treatment of differentiating myoblasts derived from the unloaded m. soleus prevented a decrease in AMPK and ACC phosphorylation, returned the expression levels of MRF and fast MyHC isoforms to the control levels, as well as maintained the expression of slow MyHC. Thus, abnormally accelerated differentiation of myoblasts isolated from the atrophied rat m. soleus can be compensated by the AICAR-assisted maintenance of control AMPK activity levels.

The structure and function of soleus muscle fibers undergo substantial remodeling under real or simulated microgravity conditions. However, unloading-induced changes in the functional activity of skeletal muscle primary myoblasts remain poorly studied. The purpose of our study was to investigate how short-term and long-term mechanical unloading would affect cultured myoblasts derived from rat soleus muscle. Mechanical unloading was simulated by rat hindlimb suspension model (HS). Myoblasts were purified from rat soleus at basal conditions and after 1, 3, 7, and 14 days of HS. Myoblasts were expanded in vitro, and the myogenic nature was confirmed by their ability to differentiate as well as by immunostaining/mRNA expression of myogenic markers. The proliferation activity at different time points after HS was analyzed, and transcriptome analysis was performed. We have shown that soleus-derived myoblasts differently respond to an early and later stage of HS. At the early stage of HS, the proliferative activity of myoblasts was slightly decreased, and processes related to myogenesis activation were downregulated. At the later stage of HS, we observed a decrease in myoblast proliferative potential and spontaneous upregulation of the pro-myogenic program.

It is well known that reduced contractile activity of the main postural soleus muscle during long-term bedrest, immobilization, hindlimb unloading, and space flight leads to increased expression of fast isoforms and decreased expression of the slow isoform of myosin heavy chain (MyHC). The signaling cascade such as HDAC4/MEF2-D pathway is well-known to take part in regulating MyHC I gene expression. Earlier, we found a significant increase of HDAC4 in myonuclei due to AMPK dephosphorylation during 24 h of hindlimb unloading via hindlimb suspension (HU) and it had a significant impact on the expression of MyHC isoforms in rat soleus causing a decrease in MyHC I(β) pre-mRNA and mRNA expression as well as MyHC IIa mRNA expression. We hypothesized that dephosphorylated HDAC4 translocates into the nuclei and can lead to a reduced expression of slow MyHC. To test this hypothesis, Wistar rats were treated with HDAC4 inhibitor (Tasquinimod) for 7 days before HU as well as during 24 h of HU. We discovered that Tasquinimod treatment prevented a decrease in pre-mRNA expression of MyHC I. Furthermore, 24 h of hindlimb suspension resulted in HDAC4 nuclear accumulation of rat soleus but Tasquinimod pretreatment prevented this accumulation. The results of the study indicate that HDAC4 after 24 h of HU had a significant impact on the precursor MyHC I mRNA expression in rat soleus.