Skeletal Muscle Microtubule Organization and Stability is Regulated by the Dystrophin-Glycoprotein Complex and Cortical Actin

Abstract

Duchenne muscular dystrophy (DMD) is a fatal X-linked myopathy caused by the loss of dystrophin in striated muscle. DMD is frequently studied in the mdx mouse which also lacks dystrophin. It has been observed by multiple independent research groups that mdx skeletal muscle presents with a disorganized cortical microtubule lattice that primarily lacks transverse microtubules as compared to the orthogonal microtubule lattice of wildtype mouse skeletal muscle. While transgenic expression of dystrophin in mdx skeletal muscle does restore microtubule organization, it is not understood how dystrophin regulates microtubule organization in vivo. This thesis implicates two regions of the dystrophin rod domain as regulators of microtubule organization and stability. Singular absence of dystrophin spectrin like repeats R4-15 or R20-24 does not impact basal microtubule organization. However, removal of both R4-15 and R20-23 from micro-dystrophin constructs results in a miniaturized dystrophin that is incapable of fully restoring microtubule organization when transgenically expressed in mdx muscle. In addition to the intermediate microtubule organization by micro-dystrophins lacking R4-15 and R20-23, we have characterized a novel microtubule pathology where transverse microtubules are lost upon eccentric contraction in the absence of either R4-15 or R20-24. Transverse microtubule loss is specific to eccentric contractions and occurs rapidly via a ROS mediated mechanism. Multiple sources of ROS appear to be involved including NOX2 but not nNOS. Moreover, loss of γ-cytoplasmic actin, β-cytoplasmic actin, or the dystrophin-glycoprotein complex (DGC) member α-dystrobrevin all cause a highly similar microtubule phenotype where transverse microtubules are lost post eccentric contraction. While both the intermediate microtubule organization and microtubule susceptibility to eccentric contraction exhibited by micro-dystrophin rescued muscle may have implications for micro-dystrophin gene therapy, the work presented in this thesis has also widened our understanding of skeletal muscle microtubule regulation to include cytoplasmic actins and DGC stability.