Dystrophin is a large protein that functions to connect the intracellular actin cytoskeleton to the sarcolemma and ultimately to the extracellular matrix via the dystrophin-glycoprotein complex. Striated muscle can lose functional dystrophin protein due to an array of mutations that result in Duchenne muscular dystrophy (DMD). As a disease with no cures, DMD is typically fatal by the third decade of life even with palliative care. One promising treatment under development for DMD is gene therapy. However, truncated dystrophin constructs are most typically utilized due to the large size of the dystrophin gene. The dystrophin deficient mdx mouse exhibits a disorganized microtubule lattice that is associated with greatly reduced ability to generate force following eccentric contraction. My preliminary data reveal that miniaturized dystrophins are not able to protect the sub-sarcolemmal microtubule lattice against the stress imposed by eccentric contraction. The objective of this study is therefore to mechanistically assess how dystrophin influences microtubules in vivo in both resting and eccentrically contracted muscle.
In aim 1, truncated dystrophins expressed on the mdx background will be utilized to define which dystrophin domains regulate microtubule stability in unstressed and eccentrically contracted muscle. Domain analysis will be paired with an iTRAQ proteomic screen to identify intermediary proteins regulating microtubule organization.
In aim 2, the physiological parameters influencing eccentric contraction induced microtubule loss will be determined as well as the dynamics of microtubule recovery over time.
The contribution of microtubule abnormalities to the pathologies associated with dystrophin-deficient forms of muscular dystrophy is not well characterized. The work proposed herein will define the dystrophin domains and intermediary proteins responsible for microtubule lattice organization as well as determine how the stabilizing interaction between dystrophin and microtubules is disrupted under the stress of muscle contraction. The study will thus provide further insight into a poorly understood muscular dystrophy pathology that likely must be addressed by any effective therapeutic.
