Altered nucleus-cytoskeleton coupling in dystrophic muscle

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Project Summary:   Duchenne muscular dystrophy (DMD), the most common and severe form of muscular dystrophy, is characterized by progressive wasting of skeletal muscles and marked susceptibility to damage. Several associated processes could underlie the pathology. The nucleus, a regulator of gene expression and a mechanotransduction hub, has increased movement in mdx (murine model of DMD) muscle. Microtubules (MTs) serve as the “railroad tracks” for cellular organelle transport, including the nucleus. The nucleus is connected to MTs and the rest of the cytoskeleton through the LINC (linkers of nucleus and cytoskeleton) complex. Both, MT organization and LINC complex expression are altered in dystrophic muscle. I will test the hypothesis that nuclear instability, due to disease-driven MT network and LINC complex alterations, results in improper myonuclear domain maintenance (with hypermobile and improperly positioned nuclei), and impaired nuclear mechanotransduction, further driving muscle weakness and susceptibility to injury in dystrophic muscle. In WT and mdx muscle I will measure: 1) nuclear spatial distribution & nuclear movement using time-lapse microscopy 2) myonuclear domain maintenance by measuring RNA spatial distribution of cargoed proteins using fluorescence in-situ hybridization; and nuclear movement & global transcriptional activity following gaps in myonuclear domain using laser ablation 3) nuclear localization of Yes- associated protein (a nuclear relay of mechanical signaling), ERK 1/2 (a key marker of muscle growth) and FRET based nuclear strain sensors, as end points of nuclear mechanotransduction, following passive stretch, isometric and eccentric contractions 4) myonuclear domain maintenance and nuclear mechanotransduction, following blockage of stretch activated channels to block sarcolemmal signaling pathways 5) the above parameters using established genetic/pharmacologic manipulations to the MT network & the LINC complex, and following mini- and micro-dystrophins that have previously shown to either fully or partially rescue MT network and susceptibility to injury Successful completion of this proposal will allow for the development of new avenues to improve musculoskeletal health for patients with DMD, and potentially other dystrophies. This proposal takes place in a multi-disciplinary environment at University of Maryland School of Medicine, with support from experts in physical therapy, physiology, molecular biology, biochemistry, and engineering, such that I can gain skills in cellular and muscle mechanics to move towards an independent, tenure-track research faculty position.