A multidisciplinary evaluation of the effectiveness of cyclosporine a in dystrophic mdx mice

Abstract

Chronic inflammation is a secondary reaction of Duchenne muscular dystrophy and may contribute to disease progression. To examine whether immunosuppressant therapies could benefit dystrophic patients, we analyzed the effects of cyclosporine A (CsA) on a dystrophic mouse model. Mdx mice were treated with 10 mg/kg of CsA for 4 to 8 weeks throughout a period of exercise on treadmill, a protocol that worsens the dystrophic condition. The CsA treatment fully prevented the 60% drop of forelimb strength induced by exercise. A significant amelioration (P < 0.05) was observed in histological profile of CsA-treated gastrocnemius muscle with reductions of nonmuscle area (20%), centronucleated fibers (12%), and degenerating area (50%) compared to untreated exercised mdx mice. Consequently, the percentage of normal fibers increased from 26 to 35% in CsA-treated mice. Decreases in creatine kinase and markers of fibrosis were also observed. By electrophysiological recordings ex vivo, we found that CsA counteracted the decrease in chloride conductance (gCl), a functional index of degeneration in diaphragm and extensor digitorum longus muscle fibers. However, electrophysiology and fura-2 calcium imaging did not show any amelioration of calcium homeostasis in extensor digitorum longus muscle fibers. No significant effect was observed on utrophin levels in diaphragm muscle. Our data show that the CsA treatment significantly normalized many functional, histological, and biochemical endpoints by acting on events that are independent or downstream of calcium homeostasis. The beneficial effect of CsA may involve different targets, reinforcing the usefulness of immunosuppressant drugs in muscular dystrophy.

Figure 1

Effects of CsA treatment on in vivo forelimb muscle strength of exercised mdx mice. Each column is the mean ± SEM from six to seven animals belonging to the different experimental groups, and in particular WT (C57/BL10), mdx (MDX), exercised mdx (Exer MDX), and exercised mdx treated with CsA (Exer MDX + CsA). Each group of bars shows from left to right the absolute forelimb strength at time 0, the absolute forelimb strength at time 4 (ie, after 4 weeks of exercise or exercise + treatment or none), and the normalized force increment. This parameter (reduced by a scale factor of 10 for graphic purposes) has been calculated by normalizing, for each mouse, the force at time 0 and the force at time 4 with respect to the relative body weights, and considering the difference between these two normalized values. The analysis of variance test for multiple comparison between groups did not show statistical difference for the absolute strength values at time 0, whereas a statistical difference was found at time 4 (F = 5.2, P = 0.00678) and for the normalized force increment values (F = 3.94, P = 0.0209). Bonferroni t-test is as follows: significantly different with respect to: a, WT values (0.0029 < P < 0.039); b, sedentary mdx mice (MDX) (0.0056 < P < 0.036); and c, untreated exercised mdx mice (Exer MDX) (P = 0.0214).

Figure 2

Morphological features of the GC muscles from exercised WT (A, B), untreated exercised mdx (C, E, G), and CsA-treated mdx mice (D, F, H), stained with toluidine blue (A, C, D, E, F) and Azan-Mallory (B, G, H). Closely packed and regularly sized myofibers (A) enveloped by a thin layer of connective tissue (B, arrow) characterize the control WT muscles. Necrotic fibers (C, arrow), and small centronucleated fibers (C, arrowhead) are present in exercised mdx muscle. The muscle fibers of untreated exercised mdx mice are also irregularly scattered and surrounded by adipose tissue (G, asterisk) or by areas of degenerating tissue with mononuclear infiltrate cells and/or small regenerating fibers (E, asterisk). CsA-treated mdx muscles showed more regular-sized centronucleated (D, arrow) and peripherally nucleated (D, arrowhead) fibers and a few connective tissues between normally packed peripherally and centronucleated myofibers (H, arrow), with less areas of mononuclear cell infiltrate (F, arrow). Scale bars: 25 μm (A, B, D–H); 28.5 μm (C).

Figure 3

Plasma creatine kinase levels measured by standard spectrophotometric analysis. Each column is the mean ± SEM from five to seven animals. Bonferroni t-test after analysis of variance (F = 3.05, P < 0.05) was significantly different with respect to sedentary mdx (*P = 0.04) and significantly different with respect to Exer mdx (**P = 0.008).

Figure 4

A: Two-site ELISA quantification of the utrophin protein levels (relative units) in the DIA muscle of WT, exercised WT (Exer WT), mdx (MDX), exercised mdx (Exer MDX), and exercised mdx mice treated with CsA (Exer MDX+CsA). Note that the value of CsA-treated group is significantly different from both sedentary and exercised WT, but it is not different from both mdx groups. The data are expressed as mean ± SD (n = 5 to 7 mice measured in quadruplicate). Statistical analysis has been performed by unpaired Student’s t-test. B: Percentage of EDL muscle fibers positive to slow type I MHC. Each bar is the mean ± SEM for 8 to 12 sections and two to three animals. Significantly different by unpaired Student’s t-test with respect to sedentary mdx (*P < 0.005) and untreated exercised mdx muscles (**P < 0.001).

Figure 5

Effect of CsA treatment on the functional cellular parameters of muscle fibers from exercised mdx mice. A: The mean values ± SEM of resting component ionic conductances to chloride (gCl) and potassium (gK) ions of EDL and DIA muscle fibers. Each column is the mean ± SEM from 29 to 67 fibers from four to five preparations for EDL and from 19 to 46 fibers from three to five preparations for DIA. The analysis of variance test for multiple comparison in EDL muscle was significant for both gCl (F = 21, P = 0.00026) and gK (F = 5.08, P = 0.03). Similarly, the analysis of variance showed significant differences in DIA for both gCl (F = 13, P = 0.0016) and gK (F = 8.25, P = 0.011). Bonferroni t-test is as follows: EDL: significantly different with respect to WT (*P < 0.025) and untreated exercised MDX mice (^§Exer MDX, P = 8.3 × 10⁻⁵). DIA: significantly different with respect to WT (*P < 0.01) and untreated exercised MDX mice (^§Exer MDX, P = 0.0031). B: The data, expressed as mean ± SEM from 15 to 30 values from three to five preparations, show the voltages for fiber contraction (MT) at each pulse duration in different experimental conditions and in particular in WT (open circles), untreated exercised mdx mice (Exer MDX, open squares), and CsA-treated exercised mdx mice (Exer MDX + CsA, open triangles). All threshold values of Exer-MDX muscle fibers were significantly more negative with respect to those of WT ones (P < 0.001). The values of Exer-MDX CsA-treated muscle fibers were slightly but not significantly less negative that those of untreated MDX ones, with the exception of the values at 5 and 10 ms (P = 0.025 and 0.05, respectively), while they remained always significantly more negative than those of WT muscle fibers (P < 0.05 and less). For some data points the SE bar is not visible being smaller than symbol size. The values have been fitted to the equation shown in the Materials and Methods section to obtain the strength-duration curves and the fitted parameters of rheobase (R) and 1/τ, described in the text. C: The intracellular free calcium concentration measured in fura-2-loaded isolated muscle fibers using calibration parameters of the Grynkiewicz’s equation determined in situ. A calibration was performed for each muscle used. Each bar is the mean ± SEM from 60 to 100 muscle fibers and three to five animals. Statistical difference between groups was determined by analysis of variance comparison (F = 30.8, P < 9.6 × 10⁻⁵) followed by Bonferroni’s t-test. *, Significantly different with respect to WT with P < 0.0001.