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Contraction-mediated damage in mdx dystrophic mouse tibialis anterior muscles is not affected by the membrane sealant poloxamer

D.R. Plant, J.G. Ryall and G.S. Lynch, Basic and Clinical Myology Laboratory, Department of Physiology, The University of Melbourne, VIC 3010, Australia.

Dystrophin deficiency causes Duchenne muscular dystrophy (DMD), a severe inherited and progressive disease of striated muscle in humans. Dystrophin is a subsarcolemmal protein responsible for linking the cytoskeleton to the extracellular matrix, and it is postulated to play a mechanical role in stabilising the muscle fibre membrane (sarcolemma) during contraction. The muscles of the mdx dystrophic mouse, an animal model for DMD, also lack dystrophin, which makes them more susceptible to contraction-induced injury (Dellorusso et al., 2002). The increased susceptibility to stretch-mediated Ca2+ overload, leading to cell contracture and death, is prevented by treatment with the membrane sealant poloxamer 188 (P-188; Yasuda et al., 2005). P-188 can incorporate into damaged membranes and effectively `plug' holes caused by lengthening contractions. We tested the hypothesis that treatment with P-188 would reduce damage and promote membrane integrity in muscles from mdx mice following contraction-induced injury.

On the day prior to experimentation, 4-6 month old mdx and wild type (C57BL/10 ScSn) mice were injected with Evans blue dye (EBD; 100 mg/kg). Mice were anaesthetised by intraperitoneal injection with pentobarbitone sodium (60 mg/kg), the right external jugular vein exposed, and a bolus dose of P-188 (460 mg/kg body mass; dissolved in 200 L sterile saline), or vehicle only, infused intravenously. The right tibialis anterior (TA) muscle was surgically exposed and the distal tendon firmly attached to the lever arm of a servomotor/transducer with the knee immobilised by a secure clamp. The right sciatic nerve was also exposed to deliver supramaximal square wave pulses via a needle electrode. The TA muscle was immersed in warmed paraffin oil to maintain temperature at 37C and maximum isometric tetanic tension (Po) recorded at the muscles optimum length in situ (intact nerve and blood supply). The muscle was subjected to two stretches of 40% strain (relative to muscle fibre length; initiated from the plateau of isometric contractions, Consolino & Brooks 2004). The magnitude of damage was assessed 5, 10 and 15 minutes later by the deficit in Po (force deficit = (Po (initial) - Po (post strain))/Po (initial)%). The TA muscle was then carefully dissected free and rapidly frozen for later cryosectioning. At the conclusion of experimentation mice were killed by cervical dislocation whilst deeply anaesthetised. Muscle cross sections (8m) were analysed using a fluorescence microscope for quantification of intracellular infiltration of EBD.

Preliminary findings indicate that force deficit was greater in mdx than wild type mice (43 9% vs 25 9%, P < 0.05), but was unaffected by P-188 treatment. The proportion of EBD positive fibres was greater in mdx than wild type mice (15 7% vs. 2 1%, P < 0.05), and was reduced in mdx mice treated with P-188 (4 2%, P < 0.05), irrespective of injury. The proportion of EBD positive fibres was not affected by the injury protocol in either wild type or mdx mice. The results indicate that P-188 does not affect the force deficit following contraction- induced injury but may play a role in maintaining sarcolemmal integrity in muscles from mdx mice, which might prevent Ca2+ overload and promote cell survival.

Consolino C.M. & Brooks, S.V. (2004) Journal of Applied Physiology, 96, 633-638.

Dellorusso C., Crawford R.W., Chamberlain, J.S., Brooks, S.V. (2002) Journal of Muscle and Cell Motility 22, 467-475. Lee R.C., River, L.P., Pan, F.S., Ji, L., Wollmann, R.L. (1992) Proceedings of the National Academy of Sciences 89, 4524-4528.

Yasuda S., Townsend, D., Michele, D.E., Favre, E.G., Day, S.M. & Metzger, J.M. (2005) Nature (in press) DOI: 10.1038/nature03844.

Supported by the Muscular Dystrophy Association (USA) and the National Health & Medical Research Council