Leupeptin-based inhibitors do not improve the mdx phenotype

Abstract

Calpain activation has been implicated in the disease pathology of Duchenne muscular dystrophy. Inhibition of calpain has been proposed as a promising therapeutic target, which could lessen the protein degradation and prevent progressive fibrosis. At the same time, there are conflicting reports as to whether elevation of calpastatin, an endogenous calpain inhibitor, alters pathology. We compared the effects of pharmacological calpain inhibition in the mdx mouse using leupeptin and a proprietary compound (C101) that linked the inhibitory portion of leupeptin to carnitine (to increase uptake into muscle). Administration of C101 for 4 wk did not improve muscle histology, function, or serum creatine kinase levels in mdx mice. Mdx mice injected daily with leupeptin (36 mg/kg) for 6 mo also failed to show improved muscle function, histology, or creatine kinase levels. Biochemical analysis revealed that leupeptin administration caused an increase in m-calpain autolysis and proteasome activity, yet calpastatin levels were similar between treated and untreated mdx mice. These data demonstrate that pharmacological inhibition of calpain is not a promising intervention for the treatment of Duchenne muscular dystrophy due to the ability of skeletal muscle to counter calpain inhibitors by increasing multiple degradative pathways.

Fig. 1.

C101 inhibits calpain activity. A range of μM C101 was added to 3 starting concentrations of calpain. In each instance, C101 reduced calpain activity.

Fig. 2.

A–D: magnification, ×20. Representative pictures of sections from EBD-treated animals: C57 control (A), C57 100 mg·kg⁻¹·day⁻¹ C101 (B), mdx (C), and mdx 100 mg·kg⁻¹·day⁻¹ C101 (D). These sections were also stained with DAPI, a nuclear stain that appears as blue. Note that the red background is far more visible in C and D (mdx) than the A and B (C57). In addition, in C and D areas of intense color can be seen indicating areas of necrosis. The % necrotic area (E) in each section was significantly increased in mdx animals compared with C57 animals. Threshold intensity was set at 100 intensity units (0–255) to identify Evan's blue dye (EBD)-positive cells. Intensity below this threshold was excluded from the sum of the necrotic area. This total area was then made relative to the section area. The background intensity (F) was increased in mdx animals, indicating a higher level of EBD absorbance into the cells, likely through sarcolemmal lesions. Median intensity units were quantified from each section. Note that regardless of group, median intensity was below the threshold of 100 intensity units used to identify the % necrotic area, indicating that the higher background intensity observed in mdx animals did not contribute to the increased necrotic area. *Significantly different from C57; †significantly different from C57 100 mg·kg⁻¹·day⁻¹ C101.

Fig. 3.

Fiber area distribution in the diaphragm following 4 wk of intraperitoneal injection of PBS or C101.

Fig. 4.

Specific tension in the diaphragm following 6 mo of daily injections of PBS or 36 mg/kg ip leupeptin (LPTN). C57 animals produced significantly more specific tension than mdx animals. Leupeptin had no effect. *Significantly different from C57 (P < 0.05); †significantly different from C57 LPTN (P < 0.05).

Fig. 5.

Representative micrographs (magnification, ×20) of trichrome-stained diaphragms from C57 (A–B) and mdx (C–D) animals. Animals were treated for 6 mo with daily injections of PBS (A, C) or 36 mg/kg LPTN (B, D).

Fig. 6.

Protease activities following 6 mo of daily PBS or 36 mg/kg LPTN injections. A: representative Western blot showing intact and cleaved talin (top) and corresponding actin blot as a loading control (bottom). Intact talin is similar between groups; however, there is a dramatic increase in cleavage in mdx animals. B: proteasome activity was increased in both sets of mdx animals compared with both C57 groups. Furthermore, LPTN caused an additional increase in proteasome activity in mdx animals. Data are means ± SE. *Significantly different from C57 (P < 0.05); †significantly different from C57 LPTN (P < 0.05); #significantly different from mdx (P < 0.05).

Fig. 7.

Representative Western blots and corresponding graphical representations of the optical densities from m-calpain (A; actin controls shown) from animals treated with LPTN or PBS for 6 mo. The μ-calpain activation remained unchanged by LPTN (B; shown with actin controls). Data are means ± SE. *Significantly different from C57 (P < 0.05); †significantly different from C57 LPTN (P < 0.05); #significantly different from mdx (P < 0.05).

Fig. 8.

Representative casein zymograms (A) and corresponding graphical representations of optical densities from μ-calpain (B) and m-calpain (C) from animals treated with LPTN or PBS for 6 mo. Data are means ± SE. *Significantly different from C57 (P < 0.05); †significantly different from C57 LPTN (P < 0.05); #significantly different from mdx (P < 0.05).