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. 2011 Jan;178(1):273-83.
doi: 10.1016/j.ajpath.2010.11.027. Epub 2010 Dec 23.

Long-term Blocking of Calcium Channels in Mdx Mice Results in Differential Effects on Heart and Skeletal Muscle

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Free PMC article

Long-term Blocking of Calcium Channels in Mdx Mice Results in Differential Effects on Heart and Skeletal Muscle

Louise H Jørgensen et al. Am J Pathol. .
Free PMC article

Abstract

The disease mechanisms underlying dystrophin-deficient muscular dystrophy are complex, involving not only muscle membrane fragility, but also dysregulated calcium homeostasis. Specifically, it has been proposed that calcium channels directly initiate a cascade of pathological events by allowing calcium ions to enter the cell. The objective of this study was to investigate the effect of chronically blocking calcium channels with the aminoglycoside antibiotic streptomycin from onset of disease in the mdx mouse model of Duchenne muscular dystrophy (DMD). Treatment in utero onwards delayed onset of dystrophic symptoms in the limb muscle of young mdx mice, but did not prevent degeneration and regeneration events occurring later in the disease course. Long-term treatment had a positive effect on limb muscle pathology, reduced fibrosis, increased sarcolemmal stability, and promoted muscle regeneration in older mice. However, streptomycin treatment did not show positive effects in diaphragm or heart muscle, and heart pathology was worsened. Thus, blocking calcium channels even before disease onset does not prevent dystrophy, making this an unlikely treatment for DMD. These findings highlight the importance of analyzing several time points throughout the life of the treated mice, as well as analyzing many tissues, to get a complete picture of treatment efficacy.

Figures

Figure 1
Figure 1
Pre-onset accumulation of calcium in dystrophin-deficient skeletal muscle suggests early implication of calcium dysregulation in DMD pathology. A: Calcium uptake (Alizarin Red positive fibers, red) in skeletal muscle from a fetus (13 weeks old) with DMD, compared with skeletal muscle from a control fetus (15 weeks old), which shows no uptake. B: Calcium uptake in tibialis anterior (TA) of C57/BL10-mdx (mdx) mice was not detectable until postnatal week 3. Central nuclei were detectable in both C57/BL10 control (BL/10) and mdx skeletal muscle (TA), confirming ongoing muscle development at this stage. Insets correspond to the higher-magnification image to the left. Scale bars = 100 μm.
Figure 2
Figure 2
Treatment with streptomycin improves central nuclei counts and pathology in TA of mdx mice in utero and 6 weeks postnatal. A: Central nuclei counts (% CN) in C57/BL10 (BL/10 C), C57/BL10-mdx control (mdx C), and streptomycin-treated (mdx S) mice. Data are expressed as means ± SD of 3–5 mice. There is a statistically significant reduction in %CN in mdx S, compared with mdx C (*P < 0.05). B: Western blot detection of TRPC3 (mol. wt. ∼97 kDa) in crude muscle extracts. Ponceau stain band mol. wt. 80–90 kDa was used as standard and loading control. We detected significantly increased TRPC3 protein in mdx skeletal muscle, compared with BL/10 control (**P < 0.01). C: Western blot results of dystrophin (mol. wt. ∼427 kDa) expression in TA from 6-week-old mdx S, mdx C, and BL/10 C. There is no dystrophin protein expressed in mdx mice, regardless of treatment, indicating that read-through of the dystrophin stop codon mutation has not occurred. Representative results from two mice per group are shown. D: Representative images of histological examination of BL/10 C, mdx C, and mdx S mice. The H&E staining shows general histology; CD45 detects all lymphocytes and is a marker of inflammation; and desmin is expressed by regenerating and newly forming fibers and is a marker for immature fibers and ongoing regeneration. Overall histology points toward less necrosis and inflammation in mdx S, compared with mdx C. TRPC3 protein expression is detectable in membrane, cytoplasm, and nuclei of both mdx C and mdx S mice and the expression appears to be increased in newly formed myotubes. This TRPC3 expression pattern in mdx control and treated mice, compared with BL/10 C, correlates with the increased expression seen on Western blots (B). Inset: Negative control image for TRPC3 immunohistochemical staining for BL/10 C. Nuclei are counterstained with DAPI in blue. Alizarin Red (ARed) staining shows necrotic foci with calcium accumulations and there is no detectable difference between mdx C and mdx S. Scale bar = 50 μm.
Figure 3
Figure 3
Continued treatment with streptomycin up to 10 weeks of age does not prevent pathological progression in TA (A–E), heart (F), and diaphragm (G). A: Central nuclei counts (% CN) in BL/10 C, mdx C, BL/10 S (C57BL/10 treated), and mdx S mice show no difference between mdx C and mdx S (P > 0.05) at 10 weeks of age. Data are expressed as means ± SD of 3–6 mice. B: Western blot detection of dystrophin (∼427 kDa) and neural cell adhesion molecule (NCAM; ∼160 kDa breakdown product) in total membrane fractions from TA muscles. Blots not run on same gels or reorganized from different parts of the same gels are all separated by a black line. C: Representative images of histological examination of TA muscles from mdx C and mdx S. Top to bottom: H&E, CD45, desmin, TRPC3, and Alizarin Red staining. Overall there appears to be no difference in pathology between mdx C and mdx S. Scale bar = 100 μm. D: TRPC3 protein expression is significantly increased in mdx C, compared with BL/10 C (*P < 0.05). Based on Western blot detection of TRPC3 (∼97 kDa). Ponceau stain band mol. wt. 80–90 kDa was used as standard and loading control. E: β-dystroglycan protein expression is significantly decreased in mdx C, compared with BL/10 C (*P < 0.05). Based on Western blot detection of β-dystroglycan (∼43 kDa). Ponceau stain band Mw 80–90 kDa was used as standard and loading control. F: Top panel: Quantification of fibrosis in 10-week-old mdx C and mdx S hearts. Data are actual measurements, expressed as mean ± SD of 3–4 mice. Bottom panel: Increased fibrosis (red) in heart muscle from mdx S, compared with mdx C, detected using Sirius Red. Scale bar = 100 μm. G: Histological analysis of diaphragm muscle from 10-week-old mice of each group using H&E shows no obvious difference in pathology between mdx C and mdx S. Scale bar = 100 μm.
Figure 4
Figure 4
Improvement of limb muscle pathology combined with aggravation of cardiac pathology after continued treatment for 6 months. A: Representative images for H&E histological examination of TA and diaphragm from mdx C and mdx S. There is an overall improvement in limb muscle pathology in 6-month-old mdx S mice, but no obvious difference in diaphragm pathology, compared with mdx C. B: Sirius Red staining detecting fibrosis (red) in TA and diaphragm from 6-month-old mdx C and mdx S confirms the H&E histological findings of improvement in limb muscle pathology observed as reduced fibrosis. No difference in diaphragm fibrosis between mdx C and mdx S. C: Examination of fibrosis in 6-month-old heart muscle of mdx C and mdx S and presence of necrotic foci (Alizarin Red) in 6-month-old heart muscle of mdx C and mdx S mice. There is increased fibrosis combined with necrosis in mdx S hearts, compared with controls, showing a worsening of the pathology. D: Top panels: Representative images of a heart MRI scan from a 6-month-old C57BL/10 control mouse showing both short-axis and long-axis views. Bottom panels: Quantification of fibrosis in 6-month-old hearts shows a statistically significant increase in fibrosis in mdx S hearts, compared with mdx C. Data are actual measurements from 6–8 mice, expressed as means ± SD. *P < 0.05. Left ventricular mass (LVM) measurements normalized to body weight performed using MRI scanning and analysis using the software program Segment (as described under Materials and Methods). There were significant differences in LVM when comparing BL/10 to all other groups using one-way analysis of variance analysis (P < 0.0001), although streptomycin had no significant effects on LVM. Data are expressed as means of 5–11 mice ± SD.
Figure 5
Figure 5
Membrane fragility is improved in limb muscle of mdx mice after 6 months treatment with streptomycin, but heart and diaphragm pathology is not improved. A: Representative images of Evans Blue Dye (EBD) uptake in TA, diaphragm, and heart tissues harvested from the same mouse for mdx C and mdx S, respectively. There is great variability in uptake both in different tissues examined from the same mouse and in the same tissue types examined from different mice. B: Quantification of EBD uptake in TA, diaphragm, and heart tissue from mdx C and mdx S. Data are from 4–5 mice, expressed as means ± SD. **P < 0.01 for EBD uptake in TA in mdx C vs. mdx S. There were no significant differences in diaphragm and heart tissue, which both show a highly variable uptake pattern. C: Western blot detection of dystrophin (∼427 kDa) expression in TA and heart muscle from 6-month-old BL/10 C, mdx C, BL/10 S, and mdx S mice. There is no dystrophin protein expressed in TA or heart muscle from mdx mice regardless of treatment, indicating that read-through of the dystrophin stop codon mutation has not occurred even after 6 months of treatment. Representative results from two mice per group are shown. D: Western blot detection of NCAM (∼160 kDa), TRPC3 (∼97 kDa), and β-dystroglycan (∼43 kDa) protein expression in total membrane fractions from 6-month-old TA muscle from BL/10 C, mdx C, BL/10 S, and mdx S. Representative results from two mice per group are shown. Ponceau staining was used as loading control (protein band of ∼80–90 kDa is shown).

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