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.

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-established that prolonged exposure to real or simulated microgravity/disuse conditions results in a significant reduction in the rate of muscle protein synthesis (PS) and loss of muscle mass. Muscle protein synthesis is largely dependent upon translational capacity (ribosome content), the regulation of which is poorly explored under conditions of mechanical unloading. Glycogen synthase kinase-3 (GSK-3) (a negative regulator of PS) is known to be activated in rat soleus muscle under unloading conditions. We hypothesized that inhibition of GSK-3 activity under disuse conditions (hindlimb suspension, HS) would reduce disuse-induced downregulation of ribosome biogenesis in rat soleus muscle. Wistar rats were randomly divided into four groups: (1) vivarium control (C), (2) vivarium control + daily injections (4 mg/kg) of AR-A014418 (GSK-3 inhibitor) for 7 days, (3) 7-day HS, (4) 7-day HS + daily injections (4 mg/kg) of AR-A014418. GSK-3beta and glycogen synthase 1 (GS-1) phosphorylation levels were measured by Western-blotting. The key markers of ribosome biogenesis were assessed via agarose gel-electrophoresis and RT-PCR. The rate of muscle PS was assessed by puromycin-based SUnSET method. As expected, 7-day HS resulted in a significant decrease in the inhibitory Ser9 GSK-3beta phosphorylation and an increase in GS-1 (Ser641) phosphorylation compared to the C group. Treatment of rats with GSK-3 inhibitor prevented HS-induced increase in GS1 (Ser641) phosphorylation, which was indicative of GSK-3 inhibition. Administration of GSK-3 inhibitor partly attenuated disuse-induced downregulation of c-Myc expression as well as decreases in the levels of 45S pre-rRNA and 18S + 28S rRNAs. These AR-A014418-induced alterations in the markers of ribosome biogenesis were paralleled with partial prevention of a decrease in the rate of muscle PS. Thus, inhibition of GSK-3 during 7-day HS is able to partially attenuate the reductions in translational capacity and the rate of PS in rat soleus muscle.

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.