Animal care and experimental protocol. The research involved male Wistar rats weighing 209 ± 21 g (mean ± SD) that were randomly divided for two HS experiments. The rats were divided into three groups for each of the experiments (n = 16/group). In each group, soleus muscles from 8 animals were collected for the measurement of passive tension with or without blebbistatin incubation. Soleus muscles from the remaining 8 animals were subjected to biochemical analysis to assess the abundance of cytoskeletal proteins.
In Experiment 1, rats were randomly assigned to the following 3 groups: 1) vivarium control (C), 2) hindlimb suspension for 7 days (7HS); 3) hindlimb suspension for 7 days with daily injections of calpain inhibitor PD150606 (7HS + PD). In Experiment 2, rats were randomly assigned to the following groups: 1) vivarium control (C), 2) hindlimb suspension for 14 days (14HS); 3) hindlimb suspension for 14 days with daily injections of calpain inhibitor PD150606 (14HS + PD). The calpain inhibitor PD150606 (Sigma-Aldrich, USA) at a dose of 3 mg/kg (diluted in 1% DMSO) was daily administered via intramuscular injections. The C and 7HS groups were treated with the equivalent amount of the vehicle.
Temperature and humidity in the vivarium room were maintained at 24°C and 50%, respectively, with 12/12 hour light/dark cycle. All rats had access to a standard diet and water ad libitum. The animals were anesthetized with an intraperitoneal injection of tribromoethanol (240 mg/kg) prior to all surgical manipulation. The animals were sacrificed with an additional tribromoethanol injection (750 mg/kg).
Hindlimb suspension. Mechanical unloading was carried out using a standard hindlimb suspension (HS) model [24]. 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. After that, the hindlimbs of the rats were lifted slightly off the floor of the cage (head-down tilt posture). The suspension height was adjusted to prevent the hindlimbs from touching any supporting surface.
In vitro muscle preparation and stimulation. Ex vivo force measurements of rat soleus muscle were carried out as previously described [37]. The isolated muscle optimal length was estimated with digital caliper in situ. Then each muscle was dissected and placed in a cooled Ringer-Krebs solution (138 mM NaCl, 5 mM KCl, 1 mM NaH2PO4, 2 mM CaCl2, 2 mM MgCl2, 24 mM NaHCO3, 11 mM glucose) with constant perfusion with carbogen (95% O2 + 5% CO2) and incubated for 15 minutes. One of the muscles was incubated for 15 min in saline with 75 µM blebbistatin (Apexbio Technology, USA), a specific inhibitor of actomyosin interactions [28]. Double knots were tied around the distal and proximal ends of the muscle near the musculotendinous junction. After that, the muscle was attached to the lever arm/force transducer from one end and to the fixed hook from the other end in the temperature-controlled (28ºC) water bath (Aurora Scientific Bath 809C). Optimal muscle length (L0) was determined with a series of twitch contractions (0.5ms, 10V). After that, the soleus muscle was set to the slack length (Ls) or the length from which the beginning of tension development was measurable. Then the muscle was stretched by 25% of Ls at a speed of 50 mm/s. The muscle was stretched for two minutes, after which the length was returned to Ls [3]. The maximum force was recorded at the end of the stretch. The tension obtained as a result of 3 repetitions for each muscle was used for all calculations. To normalize the parameters, muscle physiological cross-sectional area (CSA) was calculated as a muscle wet weight divided by the product of muscle optimal length and density [29, 17]. Force measurements were performed by using Aurora Scientific Dual Mode Lever System 305C-LR, with a data acquisition frequency of 10 kHz. Data processing was carried out by using Aurora Scientific 615A Analysis Software Suite.
Determination of the abundance of cytoskeletal proteins. Western blotting was carried out as previously described [37]. The total protein fraction was isolated and the content of desmin, α-actinin-2, α-actinin-3, and telethonin was subsequently assessed. The RIPA reagent kit (Santa Cruz, USA) was used for protein extraction. The samples were diluted in a 2X sample electrophoresis buffer (5.4 mM Tris-HCl (pH 6.8), 4%-Ds-Na, 20%-glycerin, 10%-2-mercaptoethanol, 0.02%- bromphenol blue). Electrophoresis was performed in 10% separation PAGE. Following electrophoresis, the total protein fraction was transferred to nitrocellulose membrane via western blotting. The detection of the protein of interest was performed with the following primary antibodies: desmin (Abcam, ab8592, 1:1000, USA), GAPDH (ABM, G041, USA, 1:10000), α-actinin-2 (Santa Cruz, sc-17829, USA, 1:1000), α-actinin-3 (MERCK, MABT143, USA, 1:1000) and telethonin (Abcam, ab210773, 1:1000, USA). After rinsing the membrane to remove unbound primary antibody, secondary goat anti-rabbit antibodies conjugated with horseradish peroxidase (Santa Cruz, USA) were used at a dilution of 1:50000. The blots were visualized by using the Clarity Western ECL Substrate (BioRad Laboratories, USA). Western blot data were processed by using Image Studio Digits Ver4.0 software (LI-COR Biotechnology, USA).
Electrophoresis and detection of the giant proteins (titin and nebulin) were previously described [37]. Changes in titin and nebulin contents were carried out using the technique of SDS-electrophoresis in 2.2% polyacrylamide gel with 0.5–0.6% agarose [35], with modifications aimed at improving the focusing of the studied protein bands in the gel [41]. To ensure equal loading, samples from the control and experimental groups were all run on the same gel. SDS-PAGE was performed using the Helicon VE–10 system (Moscow, Russia) at 8 mA. Following SDS-PAGE, the gels were stained with Coomassie Brilliant Blue (G-250 and R-250, 1:1). Titin and nebulin contents were normalized to the content of myosin heavy chains (MyHC).
Statistical analysis
The data are presented as mean ± standard error of the mean (SEM). Since the normal distribution of the sample was not confirmed in all cases, a nonparametric Kruskal-Wallis test was used to compare the groups with each other. The differences were accepted as statistically significant at p < 0.05.