Duchenne muscular dystrophy (DMD) is the most devastating type of muscular dystrophy, caused by a defect in the dystrophin gene resulting in progressive wasting and weakness of respiratory and locomotor muscles. By age twelve, DMD patients are no longer able to breathe or walk on their own. Muscle damage, weakness, and fiber loss (via necrosis, apoptosis) in diaphragm and limb muscles with DMD result from (a) material fatigue injury and (b) inflammation. While the link between inflammation with weakness, damage, and wasting in the diaphragm is unresolved, elevated """"""""oxidative stress"""""""" has been proposed as a mechanism. Indeed, oxidative stress is elevated and linked to muscle wasting with heart disease, lung disease, AIDS, cancer, and spaceflight. We propose that oxidative stress in the DMD diaphragm is a function of elevated NAD(P)H oxidase (NOX) with an impaired stress response of protective HSP70.
The Specific Aims of the current investigation are to (a) identify the mechanisms by which NOX oxidase leads to diaphragm weakness, apoptosis, necrosis, and wasting with DMD; and (b) determine if boosting levels of key stress proteins (e.g., HSP70) protect against oxidative stress and impaired contractile function in the dystrophic diaphragm. We hypothesize that inhibition of NOX (via apocynin) and genetic manipulation via knockout of gp91phox (regulatory subunit of NOX) will reduce oxidative stress, damage, apoptosis, necrosis, and impaired contractile function in the diaphragm. We further hypothesize that upregulation of heat shock protein70 (HSP70) by guanylguanylacetone and HSP70 overexpression will reduce oxidative stress, apoptosis, necrosis, and weakness in the diaphragm. We will use an mdx mouse model form DMD (with C57 wild types as controls) to accomplish our Specific Aims. Mice will be euthanized at 4 weeks of age, the time of peak inflammation and similar to a 3-year-old human patient. Cell protective proteins, prooxidant proteins, oxidative stress markers, indicators of apoptosis and necrosis, and damage in diaphragm samples will be assessed using Western immunoblot analysis, ELISA, and immunohistochemistry. In addition, contractile function of mdx and wild-type diaphragms will also be assessed. We anticipate that our results will have important clinical significance, as we expect to elucidate mechanisms by which oxidative stress contributes to damage, cell loss, and weakness in the dystrophic diaphragm. ? ? ? ?
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