Characterization of dysferlin deficient SJL/J mice to assess preclinical drug efficacy: fasudil exacerbates muscle disease phenotype

Abstract

The dysferlin deficient SJL/J mouse strain is commonly used to study dysferlin deficient myopathies. Therefore, we systematically evaluated behavior in relatively young (9-25 weeks) SJL/J mice and compared them to C57BL6 mice to determine which functional end points may be the most effective to use for preclinical studies in the SJL/J strain. SJL/J mice had reduced body weight, lower open field scores, higher creatine kinase levels, and less muscle force than did C57BL6 mice. Power calculations for expected effect sizes indicated that grip strength normalized to body weight and open field activity were the most sensitive indicators of functional status in SJL/J mice. Weight and open field scores of SJL/J mice deteriorated over the course of the study, indicating that progressive myopathy was ongoing even in relatively young (<6 months old) SJL/J mice. To further characterize SJL/J mice within the context of treatment, we assessed the effect of fasudil, a rho-kinase inhibitor, on disease phenotype. Fasudil was evaluated based on previous observations that Rho signaling may be overly activated as part of the inflammatory cascade in SJL/J mice. Fasudil treated SJL/J mice showed increased body weight, but decreased grip strength, horizontal activity, and soleus muscle force, compared to untreated SJL/J controls. Fasudil either improved or had no effect on these outcomes in C57BL6 mice. Fasudil also reduced the number of infiltrating macrophages/monocytes in SJL/J muscle tissue, but had no effect on muscle fiber degeneration/regeneration. These studies provide a basis for standardization of preclinical drug testing trials in the dysferlin deficient SJL/J mice, and identify measures of functional status that are potentially translatable to clinical trial outcomes. In addition, the data provide pharmacological evidence suggesting that activation of rho-kinase, at least in part, may represent a beneficial compensatory response in dysferlin deficient myopathies.

Figure 1. Maximal and specific force of extensor digitorum longus (EDL) and soleus muscles of SJL/J and C57BL6 mice:

At 25 weeks of age SJL/J (n = 5/group) and C57BL6 (n = 8/group) mice were sacrificed and in vitro muscle function tests were performed for EDL (A, C) and soleus (B, D) muscles. Shown are the values obtained for maximal force (mN) (A & B), specific force (kN/m²) (C & D). Values were compared using Student's t-test for independent samples, where * = p<0.05.

Figure 2. Effect of fasudil on extensor digitalis longorum (EDL) and soleus muscle force:

At 25 weeks of age, in vitro muscle function tests were performed on EDL (A, B, E, F) and soleus (C, D, G, H) muscles to determine the affect of fasudil treatment on muscle force in C57BL6 (B, F, D, H) and SJL/J (A, E, C, G) mice. Shown are the graphs for maximal force (MF) (mN) (A–D) and specific force (SF) (kN/m²) (E–H) for soleus and EDL of each strain. Treatment groups of each strain were compared using student's t-test for independent samples. *p = <0.05 by Student's t-test comparisons.

Figure 3. Effect of fasudil on inflammatory foci:

Representative sections of untreated (A) and fasudil treated (B) (n = 4/group) quadriceps femoris muscle of SJL/J mice sacrificed at 25 weeks of age. Frozen sections of quadriceps femoris were stained with rat anti-mouse F4/80 (Serotec, Oxford, UK) primary antibody. Anti-rat horse-radish peroxidase (DAKO, Carpinteria, CA) was used as a secondary antibody. Three non-overlapping fields of the entire tissue section were imaged (20X) and quantified. Data were graphed (mean ± SEM) and the groups were compared using student's t-test for independent samples, where *p<0.05 by Student's t-test comparison.