Dystrophin deficiency exacerbates skeletal muscle pathology in dysferlin-null mice

Abstract

Background: Mutations in the genes coding for either dystrophin or dysferlin cause distinct forms of muscular dystrophy. Dystrophin links the cytoskeleton to the sarcolemma through direct interaction with β-dystroglycan. This link extends to the extracellular matrix by β-dystroglycan's interaction with α-dystroglycan, which binds extracellular matrix proteins, including laminin α2, agrin and perlecan, that possess laminin globular domains. The absence of dystrophin disrupts this link, leading to compromised muscle sarcolemmal integrity. Dysferlin, on the other hand, plays an important role in the Ca2+-dependent membrane repair of damaged sarcolemma in skeletal muscle. Because dysferlin and dystrophin play different roles in maintaining muscle cell integrity, we hypothesized that disrupting sarcolemmal integrity with dystrophin deficiency would exacerbate the pathology in dysferlin-null mice and allow further characterization of the role of dysferlin in skeletal muscle.

Methods: To test our hypothesis, we generated dystrophin/dysferlin double-knockout (DKO) mice by breeding mdx mice with dysferlin-null mice and analyzed the effects of a combined deficiency of dysferlin and dystrophin on muscle pathology and sarcolemmal integrity.

Results: The DKO mice exhibited more severe muscle pathology than either mdx mice or dysferlin-null mice, and, importantly, the onset of the muscle pathology occurred much earlier than it did in dysferlin-deficient mice. The DKO mice showed muscle pathology of various skeletal muscles, including the mandible muscles, as well as a greater number of regenerating muscle fibers, higher serum creatine kinase levels and elevated Evans blue dye uptake into skeletal muscles. Lengthening contractions caused similar force deficits, regardless of dysferlin expression. However, the rate of force recovery within 45 minutes following lengthening contractions was hampered in DKO muscles compared to mdx muscles or dysferlin-null muscles, suggesting that dysferlin is required for the initial recovery from lengthening contraction-induced muscle injury of the dystrophin-glycoprotein complex-compromised muscles.

Conclusions: The results of our study suggest that dysferlin-mediated membrane repair helps to limit the dystrophic changes in dystrophin-deficient skeletal muscle. Dystrophin deficiency unmasks the function of dysferlin in membrane repair during lengthening contractions. Dystrophin/dysferlin-deficient mice provide a very useful model with which to evaluate the effectiveness of therapies designed to treat dysferlin deficiency.

Figure 1

Disrupted expression of dysferlin and dystrophin in skeletal muscle of dystrophin/dysferlin double-knockout mice. (A) Western blot showing the expression pattern of dysferlin (DysF), dystrophin (Dyst), β-dystroglycan (β-DG) and dihydropyridine receptor α2 (DHPRα2) in skeletal muscle tissue lysates from wild-type (WT), dysferlin-null, mdx and dystrophin/dysferlin double-knockout (DKO) mice. (B) Expression of dystrophin and dysferlin in skeletal muscles from WT, dysferlin-null, mdx and DKO mice were examined by immunofluorescence staining. (C) β-DG and sarcospan (SSPN) were greatly diminished at the sarcolemma of mdx and DKO muscles, but laminin α2 (Lam2) staining was not reduced. Scale bars: 100 μm.

Figure 2

H & E staining of masseter muscle sections from WT, dysferlin-null, mdx and DKO mice at six months of age. Scale bar: 100 μm.

Figure 3

Histopathological analyses of quadriceps muscle sections from DKO mice. (A) H & E-stained quadriceps muscle sections from dysferlin-null, mdx and DKO mice at six months of age. Scale bar: 100 μm. (B) Quantitative analysis of centrally nucleated muscle fibers (CNF) in quadriceps muscles from WT, dysferlin-null, mdx and DKO mice (n = 6 per group) at six months of age. Each group was significantly different from all the other groups. For clarity, significance is shown only for the comparisons with the DKO mice. ***P < 0.001.

Figure 4

Evans blue dye uptake analyses of quadriceps muscles and serum CK measurements. (A) Evans blue dye (EBD) fluorescence photomicrographs of quadriceps muscle sections from dysferlin-null, mdx and DKO mice at six months of age. Scale bar: 100 μm. (B) Serum creatine kinase (CK) levels were significantly different (P < 0.001) between DKO mice (n = 6) and the other groups (n = 5, 4 and 6 for WT, dysferlin-null and mdx mice, respectively). Values for mdx mice were significantly different from those for WT mice (P = 0.018). For clarity, significance is shown only for the comparisons with the DKO mice. ***P < 0.001.

Figure 5

Effects of dysferlin and dystrophin deficiencies on skeletal muscle contractile properties. We investigated the extensor digitorum longus (EDL) muscles taken from WT (n = 4), dysferlin-null (n = 6), mdx (n = 8) and DKO mice (n = 3). (A) Tetanic force measurements prior to LC. *P < 0.05 and **P < 0.01. (B) Tetanic force recovery following LC. Dysferlin deficiency had a significant effect on force recovery in the absence of dystrophin, as indicated by a significant interaction (P = 0.04) between dysferlin, dystrophin and time after LC on the basis of analysis of variance. (C) Summary of estimated maximal recovery levels following LC. *P < 0.05. (D) Summary of force recovery rate following LC. *P < 0.05.