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Muscle-specific heat shock protein 72 (HSP72) overexpression improves muscle structure and function in dystrophic mdx mice

S.M. Gehrig,1 T.A. Sayer,1 C. van der Poel,1 D. Henstridge,2 J.D. Schertzer,1 J.E. Church,1 M.A. Febbraio2 and G.S. Lynch,1 1Basic and Clinical Myology Laboratory, Department of Physiology, The University of Melbourne, VIC 3010, Australia and 2Cellular and Molecular Metabolism Laboratory, Baker IDI, PO Box 6492, St Kilda Road Central, VIC 8008, Australia.

Duchenne muscular dystrophy (DMD) is the most severe of the muscular dystrophies, affecting 1 in 3,500 live male births. Affected patients generally die in their twenties, with respiratory and/or cardiac failure ultimately causing death in most cases (Finsterer, 2006). Absence of the dystrophin protein results in muscle fibre fragility, whereby contractions result in membrane tears and Ca2+ influx. Coupled with abnormalities in intracellular Ca2+ handling, this results in an elevated cytosolic [Ca2+], resulting in the subsequent activation of degenerative pathways. Chronic muscle fibre degeneration and increasingly ineffective regeneration results in fibrotic tissue infiltration leading to major functional impairments in DMD patients. Heat shock protein 72 (HSP72) has been shown to protect contractile function and improve calcium handling dynamics under conditions of stress in cardiac muscle (Kim et al., 2006). We tested the hypothesis that HSP72 overexpression would ameliorate the dystrophic pathology and thus preserve muscle function in mdx dystrophic mice.

Female mdx mice were crossed with male mice expressing a rat inducible HSP72 transgene under the control of a chicken β-actin promoter, which limited transgene expression to skeletal and cardiac muscle (and brain) tissue (Marber et al., 1995). F1 generation males were mated with female mdx mice to yield an equal proportion of mdxHSP72 and mdx littermate controls. Mice (25-30 week old) were anaesthetised (60 mg/kg sodium pentobarbitone), and the functional properties of diaphragm muscle strips were measured in vitro as described previously (Lynch et al., 1997). Mice were killed by diaphragm and cardiac excision while still anaesthetized deeply. Diaphragm muscle strips were also frozen for subsequent histological analysis. Blood was sampled to measure serum creatine kinase (CK) levels, a myoplasmic protein commonly used as a measure of whole body muscle breakdown. In a separate group of mice, Evans blue dye (EBD) was injected (1% w/v, 10μl/g BM, i.p.) for assessment of damaged and necrotic muscle fibres.

HSP72 protein expression was elevated significantly in the muscles of mdxHSP72 compared with mdx littermate control mice. HSP72 overexpression improved specific (normalised) force in isolated diaphragm muscle strips (p < 0.05), reduced collagen infiltration (p < 0.05) and reduced minimal Ferets variance coefficient (used as an index of the severity of the pathology; p < 0.05). Serum CK levels were significantly lower in mdxHSP72 compared with mdx littermate controls (p < 0.05), which was further supported by a reduction in EBD-positive fibres indicating fewer damaged and/or necrotic fibres (p < 0.05).

Overexpression of HSP72 improved the dystrophic skeletal muscle pathology in mdx mice, especially in the severely affected diaphragm muscle. Further research is required to determine the therapeutic potential of this novel approach for DMD and related conditions.

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Kim Y-K, Suarez J, Hu Y, McDonough PM, Boer C, Dix DJ, Dillman WH. (2006) Circulation 113: 2589-2597.

Lynch GS, Rafael JA., Hinkle RT, Cole NM, Chamberlain JS, & Faulkner JA. (1997) American Journal of Physiology. Cell Physiology 272: 2063-2068.

Marber MS, Mestril R, Chi S-H, Sayen R, Yellon DM, Dillman WH (1995) The Journal of Clinical Investigation 95: 1446-1456.