The animals used in the study were male rats of the Wistar strain (Scanbur BK AS Sollentuna, Sweden) that were 24 weeks old at the beginning of the experiment.

The rats were assigned to control (Contr) (n = 8) and dexamethasone (Dex) (n = 8) groups. Dexamethasone (Dexafort 3 mg/ml; International B.V. Boxmeer, The Netherlands) was diluted to 200 µg/ml with 0.15 M NaCl and administered intraperitoneally at 100 µg/100 g body weight (bw) daily for 10 days. The control animals received appropriate amounts of 0.15 M NaCl. The animals were anaesthetised by intraperitoneal injection of ketamine (2.5 mg/100 g bw) (Calysol, Gedeon Richter A.O. Budapest, Hungary) and diazepam (2.5 mg/100 g bw) (Lab Renaudin, France). The experimental protocol was approved by the Animal Experimentation Committee at the Estonian Ministry of Agriculture.

The central part of the muscle belly of the gastrocnemius muscle (Gas) of the anaesthetised rats was tested five times with a pause of 1 s. The testing end of the device was placed on the surface of the skin above the peripheral muscle under investigation. The electromagnet contained in the device produces a short constant force impulse, which is transmitted via the testing end to the contact area. The impulse terminates in a quick release, and this mechanical influence induces damped natural oscillation in the muscle under investigation [15]. This oscillation is picked up by the acceleration transducer situated on the testing end of the device. The acceleration of the first period of the oscillation, which is calculated from the oscillation graph, characterises the deformation of the muscle caused by the testing end. The acceleration is used to calculate the stiffness of the muscle (C), according to the following formulae: C = m·a_max/Dl, where m is the mass of the testing end of myometer; a_max is the maximal amplitude of oscillation, and Δl is the depth of the displacement of the testing end. The period of the natural oscillation (T [seconds]) that is performed by the oscillating muscle mass together with the mass of the testing end can be determined, and the frequency of oscillation of the muscle (f [Hz]) is calculated: f = 1/T [Hz]. The quantity of mechanical energy of elasticity dissipated during the first period of oscillation is characterised by the logarithmic decrement of damping (Q).The logarithmic decrement of damping was calculated according to the following formulae: Q = ln(a₃/a₅), where Q is the oscillation logarithmic decrement of the tissue; a₃ and a₅-the oscillation amplitudes. The natural frequency of oscillation of the muscle at relaxation characterises the tone of the muscle and describes the development of force by the muscle at contraction. The decrement characterises muscle elasticity, i.e. the ability of the muscle to restore its initial shape after contraction. The stiffness describes the ability of the muscle to resist the changes in its shape that are caused by external forces. Previous studies have demonstrated that myometrical measurements in human skeletal muscle show good reliability, validity, and repeatability [16-18].

Hind-limb grip strength was measured before and after 10 days of dexamethasone treatment using a Grip Strength Meter 0167-004L (Columbus Instruments, USA).

The extensor digitorum longus (EDL) and Gas muscles were dissected, trimmed of visible fat and connective tissue, weighed, frozen in liquid nitrogen, and stored at −80˚C until further processing.

The titin, nebulin and MHC content of muscle samples was determined by gradient 2.8% - 12% SDS-PAGE according to the previously described method [10] with slight modifications. We reduced acrylamide content of the separating gel to 2.8% and used the buffer solution that was used to saturate the gel at pH 9.0 instead of the standard pH 8.6 - 8.8 [19]. The muscle samples were prepared as described previously [10]. Frozen muscles were pulverized to a fine powder in liquid nitrogen and tissue powders were homogenized in ice-cold lysis buffer (5 mM ethylenediaminetetraacetic acid, EDTA; 30 µg/ml leupeptin; 100 µg/ml phenylethylsulfonyl fluoride, PMSF; and 20 mM Tris; pH 7.5) on ice. We used the same weight to volume dilution (1 mg muscle tissue/15 µl of lysis buffer). Also, the total content of protein in the myofibrils was measured with a protein assay kit (BioRad, Hercules, California). Subsequently, myofibrils were solubilized with an equal volume of 2X SDS sample buffer (2 mM EDTA, 2% SDS, 1.2 M β-mercaptoethanol, 20% glycerol, 0.02% bromophenol blue, and 20 mM Tris; pH 8.0) and heated to 50˚C for 20 min [10].

The gel slab was stained with PageBlue^TM Protein Staining Solution (Fermentas, Canada), and the protein bands were analysed densitometrically using the Image Master^® 1D Program, Version 3.0 (Amersham Pharmacia Biotech, UK).

The optical density integrals (ODI) of total titin (T1 + T2), nebulin and total MHC, were measured for each loading volume, and the slope of the relationship between the ODI and loading volume was determined by linear regression analysis. The ratios of titin/MHC, nebulin/MHC, and nebulin/titin were calculated.

MHC isoforms proportion was determined by 8% SDSPAGE [20]. According to Kohn and Myburgh [21], the reducing agent (b-mercaptoethanol) was added to the running buffer in the upper chamber.

Muscle samples were prepared for MHC isoforms electrophoresis as described previously. Frozen muscles were thawed on ice, cut into small pieces, and washed with five volumes 20 mM NaCl, 5 mM sodium phosphate, 1 mM EGTA (pH 6.5). Myofibrillar protein was extracted with three volumes 100 mM sodium pyrophosphate, 5 mM EGTA, 1 mM dithiothreitol (pH 8.5) diluted with glycerol 1:1 (vol/vol) and were stored at –80˚C until analysis. Myofibrils containing 0.5 µg of protein were loaded on the gel after being incubated for 10 min at 65˚C in sample buffer containing 62.5 mM Tris-HCl, pH 6.8, 20% (vol/vol) glycerol, 5% (vol/vol) β-mercaptoethanol, 2.0% SDS, 0.05% bromphenol blue [22].

All gels were stained with silver [23], scanned, and percentage distribution of MHC isoforms was determined using a computer-based image analysis system and software (Image Master® 1D Program, Version 3.0, Amersham Pharmacia Biotech, UK).

Descriptive values are presented as means (standard error (SE). The Pearson correlation coefficient was used to quantify association. The two-sample t-test was conducted to determine differences in the proportion of titin between muscles from the Dex and Contr groups. Linear regression models of ODI and loading volume were estimated to compare the Dex and Contr groups with respect to the ratio of the slopes. In addition, the ratios of the ODI values (titin/MHC, nebulin/MHC) were determined to analyse the differences between the two groups. The significance level used in the analyses was 0.05.

After 10 days of dexamethasone treatment, the wet weight and hind-limb grip strength of Dex group was significantly lower than that in the age-matched Contr group (Table 1). The wet weight of EDL muscle was 30.2% lower and in Gas muscle 40.6% lower in Dex group than in Contr group. The hind-limb grip strength was 51% lower in Dex group as compared to Contr group.

After 10 days of dexamethasone infusion, muscle viscoelastic properties of Dex group were significantly different from Contr group (Table 2).

Elasticity is the ability of the muscle to recover its shape, characterised by the logarithmic decrement of the damped oscillations. The smaller the value of decrement, the more elastic is the tissue. The decrement was significantly higher in the atrophied Gas muscle in Dex group than in Contr group. In addition, the value of decrement correlated positively with the degree of atrophy (r = 0.79

Table 1. Body weight, muscle wet weight and hind-limb grip strength of Dex and Contr group. Values are means (SE).

^**Significant difference (P < 0.001) between groups.

Table 2. Viscoelastic properties of Dex and Contr group. Values are means (SE).

^*Significant difference (P < 0.05) between groups.

P < 0.05) (Figure 1).

Stiffness reflects the resistance of the tissue to the force that changes its shape. In Dex group, stiffness was significantly lower than in Contr group.

Tissue tone is a biomechanical property of the tissue, which can be characterised by two main parameters: stiffness and elasticity. A higher oscillation frequency indicated higher tone. The tone was significantly higher in Dex group than in Contr group.

Titin, nebulin and total MHC were separated using the 2.8% - 12% SDS-PAGE minigel system. No significant changes were seen in the titin isoforms (T1 and T2) composition after dexamethasone treatment compared to Contr group.

The relationship between loading volume and ODI for titin, nebulin, and MHC was determined by linear regression analysis. The slopes ratio of the ODI/loading volume curve (Dex/Cont) for MHC decreased by 17.7%, while the magnitude was more pronounced for titin and nebulin (23.6% and 29.4% respectively).

The densitometric gel analysis revealed differences in the titin to MHC ratio (titin/MHC), nebulin to MHC ratio (nebulin/MHC) and nebulin to titin ratio (nebulin/titin) between the groups (Figure 2). The ratio of titin/MHC decreased by 8.61%, the ratios of nebulin/MHC and nebulin/titin also decreased significantly by 30.3% (P < 0.05) and 28.9% (P < 0.01), respectively.

MHC isoforms composition of the EDL muscle was determined by 8% SDS-PAGE. We observed significant differences in all MHC isoforms between groups (Table 3). The mean proportion of MHC I (slow) was higher in Dex group and the proportion of fast type MHC isoforms was lower in Dex group than in Contr group. These changes in MHC isoforms proportion showed fast to slow MHC isoforms transition. There was also a relationship between atrophy of the muscle and the relative

content of MHC IIb isoform in this muscle (Figure 3).