Effects of Nandrolone in the counteraction of skeletal muscle atrophy in a mouse model of muscle disuse: molecular biology and functional evaluation

Muscle disuse produces severe atrophy and a slow-to-fast phenotype transition in the postural Soleus (Sol) muscle of rodents. Antioxidants, amino-acids and growth factors were ineffective to ameliorate muscle atrophy. Here we evaluate the effects of nandrolone (ND), an anabolic steroid, on mouse skeletal muscle atrophy induced by hindlimb unloading (HU). Mice were pre-treated for 2-weeks before HU and during the 2-weeks of HU. Muscle weight and total protein content were reduced in HU mice and a restoration of these parameters was found in ND-treated HU mice. The analysis of gene expression by real-time PCR demonstrates an increase of MuRF-1 during HU but minor involvement of other catabolic pathways. However, ND did not affect MuRF-1 expression. The evaluation of anabolic pathways showed no change in mTOR and eIF2-kinase mRNA expression, but the protein expression of the eukaryotic initiation factor eIF2 was reduced during HU and restored by ND. Moreover we found an involvement of regenerative pathways, since the increase of MyoD observed after HU suggests the promotion of myogenic stem cell differentiation in response to atrophy. At the same time, Notch-1 expression was down-regulated. Interestingly, the ND treatment prevented changes in MyoD and Notch-1 expression. On the contrary, there was no evidence for an effect of ND on the change of muscle phenotype induced by HU, since no effect of treatment was observed on the resting gCl, restCa and contractile properties in Sol muscle. Accordingly, PGC1α and myosin heavy chain expression, indexes of the phenotype transition, were not restored in ND-treated HU mice. We hypothesize that ND is unable to directly affect the phenotype transition when the specialized motor unit firing pattern of stimulation is lacking. Nevertheless, through stimulation of protein synthesis, ND preserves protein content and muscle weight, which may result advantageous to the affected skeletal muscle for functional recovery

Effect of SMT C1100 on <i>in vivo</i> parameters of exercised <i>mdx</i> mice.

<p>(A) Maximal fore limb strength after 4 weeks of either exercise and/or drug treatment. The values are mean ± SEM from the number of animals shown in each bar (B) Normalized force increment, i.e. difference between the mean values of normalized fore limb strength at time 4 and at time 0. Normalized force values have been calculated for each mouse as the ratio of maximal fore limb strength to mouse body weight. The values are mean ± SE. The SE of ΔNF has been calculated as detailed elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019189#pone.0019189-DeLuca1" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019189#pone.0019189-DeLuca2" target="_blank">[21]</a>. For (A) and (B) statistical significance between groups was evaluated by ANOVA test for multiple comparison (F-values) and Bonferroni t-test <i>post hoc</i> correction. Significantly different versus <b>*</b>sedentary mdx and ^§Exer <i>mdx</i>; p<0.05; (C) Resistance to treadmill running, calculated as the maximal distance the mouse can run when undergoing a single bout of treadmill exercise. The values are mean ± SEM from 3–7 mice and show the maximal distance run (in meters) at T0 (start of forced exercise and dosing) and after four (T4) and five weeks (T5). Statistical significance between groups was evaluated by ANOVA test for multiple comparison (F-values) and Bonferroni t-test <i>post hoc</i> correction. Highly significant differences were observed between groups and within groups at the different ages (F>10; p<0.005). The symbols show statistical differences *versus sedentary <i>mdx</i> at T4 and #versus vehicle-treated exercised <i>mdx</i> at either T4 or T5 (p<0.05 and less).</p

Reduction in secondary pathological features.

<p>(A) Data demonstrates the reduction in overall skeletal muscle inflammation and fibrosis from <i>mdx</i> treated with SMT C1100 compared to vehicle only. SMT C1100 (50 mg/kg) or vehicle was delivered daily by oral gavage to groups of six <i>mdx</i> mice aged around 17 d for a total of 28 days. The TA, EDL, soleus, and diaphragm were removed and five sections from each muscle were stained and analysed blind by a board-certified veterinary pathologist for evidence of inflammation and fibrosis using a standard pathology scoring method described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019189#s2" target="_blank">methods</a> section. Scoring (0–3) was made for each section from each muscle then averaged for all muscles to give an overall assessment of improvement in the pathological effects of dystrophin deficiency; (B) Qualitative assessment of EDL muscle from SMT C1100-dosed <i>mdx</i> scored as 1 = mild - occasional mononuclear inflammatory cells in the inter-bundle connective tissue with focal aggregations of mononuclear inflammatory cells. The arrows mark foci of inflammation. Qualitative assessment of EDL dosed with vehicle and scored as 2 = moderate - multiple foci of mononuclear inflammatory cell infiltration in the inter-bundle connective tissue; occasional mononuclear inflammatory cells between individual muscle fibres.</p

<i>Ex vivo</i> analysis of SMT C1100 activity in the <i>mdx</i> mouse.

<p>(A) <i>mdx</i> mice were treated with SMT C1100 (50 mg/kg/day) or vehicle only (0.1% Tween-20/5% DMSO in PBS) via daily i.p. injection from two weeks of age for four weeks. Whilst contracting tetanically, EDL muscles were stretched at 15% of their fibre length. The difference in force produced between the first and fifth stretch is represented as an indicator of the resistance of the muscle to stretch-induced damage. *p<0.05; **p<1.0×10⁻⁵; (B) levels of serum creatine kinase following oral gavage of <i>mdx</i> mice with 50 mg/kg SMT C1100 or vehicle from three weeks of age for four weeks (C) Muscles from the dosing described in (B) were processed to assess the percentage of centrally nucleated fibres *p<0.01; (C)***p = 0.0001; **p = 0.005; *p = 0.003.</p

<i>In vitro</i> activity of SMT C1100.

<p>(A) SMT C1100 dose response in murine <i>H2k-mdx</i> utrnA-luc cells expressing the human utrophin promoter linked to a luciferase reporter gene. Cells were treated with compound for 48 h in standard growth medium containing 0.3% DMSO. The chart shows relative luminescence (RLU) in relation to five different doses of SMT C1100. A Four Parameter Logistic Model was used to generate an EC₅₀. Points represent a mean ±S.E. of three experiments and are typical of the results for all batches of SMTC1100. The structure for SMTC1100 is shown; (B) SMT C1100 significantly increased mRNA copy number of the utrophin transcript in SkMC cells. In this assay Gene Expression Assay 4326315 was used for β-actin detection and Gene Expression Assay Hu01125984_m1 was used for utrophin transcript detection (both Applied Biosystems). Cells were exposed to SMT C1100 in standard media with 1% DMSO (vehicle) for 72 hours with six biological replicates. *p = 0.01 relative to vehicle only; (C) Utrophin protein levels in human DMD cell line treated with SMT C1100 (1 µM) or vehicle (0.1% DMSO). Blots were stained with anti-utrophin (MANCHO3; 1∶100) and ECL HRP-conjugated anti-mouse antibody (GE Healthcare). Bands were quantified using Image J and arbitrary units represent utrophin levels corrected for equal loading by α-actinin immunostaining. Results represent a mean ± S.E based on n = 3. †p = 0.00683; §p<0.001; #p<0.005 relative to vehicle-treated cells.</p