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Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder which leads to early death (Bonilla et al., 1988; Hoffman, Brown, & Kunkel, 1987). The mdx mouse, which is an established animal model for DMD, similarly lacks dystrophin and displays a peak in muscle cell necrosis at approximately 3-4 weeks of age, however in direct contrast to DMD, mdx mice experience muscle regeneration into adulthood (>6 weeks) (Bonilla et al., 1988; Cullen & Jaros, 1988; Spencer, Croall, & Tidball, 1995). The amino acid taurine has been shown to increase muscle strength in peak damage 28 day mdx mice (Barker, Horvath, van der Poel, & Murphy, 2017). Matrix metalloproteinase 2 (MMP2) and MMP9 reportedly play a critical role in differentiation and regeneration of skeletal muscle fibres through processing extracellular substrates (Chen & Li, 2009) and MMP9, but not MMP2, has been implicated in the pathology of the mdx mouse. In this study we examined the gelatinolytic activity and abundance of MMP2 and MMP9 in 28 day (D28) and D70 wild-type (WT) and mdx mice, and in D28 mdx mice with or without pre-natal taurine (tau) supplementation.
All procedures were approved by La Trobe University Animal Ethics Committee. Male mdx and WT mice (C57/BL10ScS) (D28 and D70) were anesthetized with an intraperitoneal injection of 10 μL/g Nembutal (Sodium Pentobarbitone) prior to euthanasia by heart excision, then gastrocnemius muscle was collected. Muscle samples from WT and mdx mice at D28 and D70, and mdx taurine mice (D28 mdx tau) were analysed using both zymography (to determine gelatinolytic activity of both pro and active forms of MMPs) and Western blotting (to determine total MMP protein contents).
Summaries of the results are shown in the Tables.
Table A.
Protein abundance and activity of pro- and active-MMP2 and proMMP9 in
D28 and D70, WT and mdx mice, by zymography and Western blotting.
Mean ± SD, *p<0.05
compared to D70 WT, #p<0.05 compared to D28 mdx mice,
one way ANOVA, Holm-Sidak’s multiple post-hoc test.
| ProMMP2 | ActiveMMP2 | ProMMP9 | |||
| Zymography | Western | Zymography | Zymography | Western | |
| D28 WT (n = 5) | 1.2 ± 0.6 * | 1.4 ± 0.6 * # | 0.5 ± 0.4 * # | 1.7 ± 1.6 * | 1.6 ± 0.6 * |
| D70 WT (n = 6) | 0.2 ± 0.1 | 0.3 ± 0.2 | 0.05 ± 0.02 | 0.4 ± 0.4 | 1.0 ± 0.2 |
| D28 mdx (n = 6) | 1.0 ± 0.5 | 2.4 ± 0.4 | 1.0 ± 0.4 | 1.0 ± 0.4 | 1.3 ± 0.2 |
| D70 mdx (n = 6) | 0.6 ± 0.3 * | 1.5 ± 0.4 * # | 0.4 ± 0.2 * # | 0.5 ± 0.2 | 1.0 ± 0.3 |
| D28 mdx with or without Taurine (Tau) - Zymography | |||
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ProMMP2 | ActiveMMP2 | ProMMP9 |
| − Tau (n = 5) | 1.0 ± 0.3 | 1.0 ± 0.4 | 1.0 ± 0.1 |
| + Tau (n = 5) | 0.8 ± 0.2 | 0.5 ± 0.1 # | 0.8 ± 0.2 # |
The data indicated that the amount of MMP2 is greater in the necrotic peak phase (D28) of mdx mouse (Table A), suggesting that MMP2 may play an important role in the necrosis of skeletal muscle fibres in muscular dystrophy. This is supported by the observation that the improved muscle function seen with Tau supplementation was accompanied by a decrease in the gelatinolytic activity of active MMP2 (Table B). MMP9 showed age-related differences in WT mice, but not in mdx mice, suggesting that it may play a role during the development of skeletal muscle but is not crucial for the degeneration / regeneration cycles occurring in the mdx mouse model.
Barker RG, Horvath D, van der Poel C, Murphy RM. (2017). PLoS currents doi: 10.1371/currents.md.9a3e357a0154d01050b591601cbd4fdb
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Cullen MJ, Jaros E. (1988). Acta Neuropathol 77, 69-81.
Hoffman EP, Brown RH, Jr., Kunkel LM. (1987). Cell 51, 919-928.
Spencer MJ, Croall DE, Tidball JG. (1995). J Biol Chem 270, 10909-10914.