Reduced Sarcolemmal Membrane Repair Exacerbates Striated Muscle Pathology in a Mouse Model of Duchenne Muscular Dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is a common X-linked degenerative muscle disorder that involves mutations in the DMD gene that frequently reduce the expression of the dystrophin protein, compromising the structural integrity of the sarcolemmal membrane and leaving it vulnerable to injury during cycles of muscle contraction and relaxation. This results in an increased frequency of sarcolemma disruptions that can compromise the barrier function of the membrane and lead to death of the myocyte. Sarcolemmal membrane repair processes can potentially compensate for increased membrane disruptions in DMD myocytes. Previous studies demonstrated that TRIM72, a muscle-enriched tripartite motif (TRIM) family protein also known as mitsugumin 53 (MG53), is a component of the cell membrane repair machinery in striated muscle. To test the importance of membrane repair in striated muscle in compensating for the membrane fragility in DMD, we crossed TRIM72/MG53 knockout mice into the mdx mouse model of DMD. These double knockout (DKO) mice showed compromised sarcolemmal membrane integrity compared to mdx mice, as measured by immunoglobulin G staining and ex vivo muscle laser microscopy wounding assays. We also found a significant decrease in muscle ex vivo contractile function as compared to mdx mice at both 6 weeks and 1.5 years of age. As the DKO mice aged, they developed more extensive fibrosis in skeletal muscles compared to mdx. Our findings indicate that TRIM72/MG53-mediated membrane repair can partially compensate for the sarcolemmal fragility associated with DMD and that the loss of membrane repair results in increased pathology in the DKO mice.

Keywords: dystrophy; fibrosis; membrane repair; muscle; sarcolemma.

Figure 1

Membrane repair and integrity is compromised in DKO mice. (A) Representative images of IgG staining. Paraffin sections of EDL and TA muscles stained with fluorescent anti-mouse-IgG antibodies demonstrate the distribution of IgG-positive and IgG-negative myocytes in selected skeletal muscles. Quantification analysis of IgG-positive myocytes for the EDL and TA show a significant increase in positive muscle myocyte damage in the DKO group. EDL n = 4, p = 0.8456; TA n = 4, p = 0.0081. (B) Representative images of FM4-64 dye in whole FDB muscles from mdx and DKO mice. The area under the curve (AUC) of FM4-64 fluorescence traces displays different membrane resealing in DKO mice. mdx n = 22 myocytes; DKO n = 16 myocytes, p = 0.0052. ** = p < 0.01. Data represented as means ± SEM. (A) Scale bar = 50 µm, (B) Scale bar = 20 µm.

Figure 2

Membrane repair proteins are altered in DKO mice. (A) Soleus and (B) EDL muscle lysates from 6-week-old mice were used for Western blotting to detect changes in membrane repair proteins. Cav-3 protein expression increases in both muscles; while dysferlin levels are only elevated in EDL muscles. Statistical analysis was performed with an unpaired two-tailed t-test assuming unequal variances, and with Welch’s t-test for unbalanced designs. (A) (n = 4) Dysf p = 0.9177, Cav-3 p = 0.0339; (B) (n = 4) Dysf p = 0.0212, Cav-3 p = 0.0458. * = p < 0.05. Data represented as means ± SEM.

Figure 3

Histological analysis of skeletal muscle of mdx and DKO mice at 6 weeks. H&E staining of EDL (A), soleus (B), and tibialis anterior (C) sections from mdx and DKO mice at six weeks of age was analyzed for frequency distribution of myocyte cross-sectional area (CSA) and central nuclei. There was no difference in the histology of any of the muscles (n = 3) for all groups tested. (D) Maximal force (F-max) of ex vivo EDL and soleus muscles from mdx and DKO mice. Force was significantly reduced in the DKO EDL muscles. Differences in F-max were compared by unpaired two-tailed t-test assuming unequal variances, and with Welch’s t-test for unbalanced designs. F-max EDL: mdx n = 10, DKO n = 13 p = 0.0414; Soleus: mdx n = 12, DKO n = 7, p = 0.2532. * = p < 0.05. Data represented as means ± SEM to indicate the confidence level in the mean at each frequency. Scale bar = 100 µm.

Figure 4

Histology and functional analysis of skeletal muscle of mdx and DKO mice at 1.5 years. H&E staining of EDL (A), soleus (B), TA (C) sections from mdx and DKO mice at 1.5 years of age was analyzed for frequency distribution of myocyte cross-sectional area (CSA) and central nuclei, n = 4 per group (D). Maximal force of ex vivo EDL and soleus muscles from mdx and DKO mice. EDL mdx n = 11, DKO n = 9, p = 0.0150; Soleus mdx n = 10, DKO n = 9, p = 0.8944. CSA was analyzed with two-way ANOVA. Differences in central nuclei counts and maximal force were tested for statistical significance with an unpaired two-tailed t-test assuming unequal variances, and with Welch’s t-test for unbalanced designs. * = p < 0.05. Data represented as means ± SEM. Scale bars = 100 µm.

Figure 5

Masson’s trichrome stain analysis implicates robust fibrosis of DKO mice at 1.5 years. DKO mice experience repeated bouts of damage throughout their lifetime, leading to increased fibrotic tissue in skeletal (A–C) muscle. Data represented as means ± SEM. n = 5 for all groups. EDL p = 0.0216, Soleus p = 0.0490, TA p = 0.0280. * = p < 0.05. (A,B) Scale bar = 50 µm, (C) Scale bar = 100 µm.

Figure 6

Membrane repair and integrity is compromised in aged DKO mice. (A) Representative images of IgG staining. Paraffin sections of EDL, soleus, and TA muscle stained with fluorescent anti-mouse-IgG antibodies demonstrate the distribution of IgG-positive and IgG-negative myocytes in selected skeletal muscles. Quantification analysis of IgG-positive myocytes for the EDL, soleus, and TA show a significant increase in positive muscle myocyte damage in the DKO group. Data analyzed by unpaired two-tailed t-test assuming unequal variances, with Welch’s t-test for unbalanced designs. n = 4 for all groups, EDL p = 0.0192, Soleus p = 0.0221, TA p = 0.0399. (B) Representative images and time-dependent accumulation of FM4-64 dye in whole FDB muscles from mdx and DKO mice. Laser injury was induced at time 0 on the time course graph. The area under the curve (AUC) of FM4-64 fluorescence traces displays different membrane resealing in DKO mice. AUC was analyzed with a t-test. mdx n = 10, DKO n = 9, p = 0.0046. * = p < 0.05, ** = p < 0.01. Data represented as means ± SEM, (A,B), Scale bar = 100 µm, (C) Scale bar = 200 µm, (D) Scale bar = 20 µm.