Increased YAP nuclear signaling in dystrophic muscle


Topic:

Pre-Clinical Research

Poster Number: 14

Author(s):

Shama Iyer PhD, Sameer Shah PhD, Christopher Ward PhD, Joseph Stains PhD, Richard Lovering PT, PhD

Institutions:

1. University of Maryland, School of Medicine, 2. University of California San Diego, 3. University of Maryland School of Medicine, 4. University of Maryland School of Medicine, 5. University of Maryland School of Medicine

Skeletal muscle experiences mechanical forces that regulate its development, hypertrophy, and homeostasis. The absence of dystrophin underlies the pathology in Duchenne muscular dystrophy, although the role of altered mechanotransduction in the pathology of muscle weakness and injury remains unclear. YAP (Yes-associated protein) serves as an indicator of nuclear mechanical signals and can induce a wide range of downstream signaling cascades. We tested the hypothesis that mdx (dystrophin-null) mice have reduced YAP signaling that contributes to the dystrophic pathology. Following in-vivo contractile loading in healthy (wild type, WT) and mdx mice, we performed western blots of whole and fractionated muscle homogenates to examine the ratio of phospho (cytoplasmic) YAP to total YAP and nuclear YAP, respectively. With contractile loading, nuclear translocation of YAP was increased in WT. Surprisingly, without contractile loading, mdx muscles showed higher YAP expression that was proportionally less phosphorylated suggesting higher basal YAP signaling than in WT. Contractile loading did not further increase nuclear YAP translocation in mdx muscles. In-vitro assays of isolated myofibers plated on substrates with high stiffness showed YAP nuclear labeling for both genotypes, indicating dystrophin’s absence does not alter the machinery required for nuclear translocation. We also found increased gene expression of YAP downstream targets, supporting the finding of hyperactive YAP in mdx muscles. Pharmacological inhibition of YAP activity resulted in decreased fibrosis in-vivo, indicated by reduced gene expression of vimentin. We conclude that nuclei in mdx muscles have heightened YAP signaling, whereby constitutively active YAP results in a failure to respond to load. Alternatively, the heightened YAP signaling in mdx nuclei could be attributed to the increased state of “pre-stress” with increased cytoskeletal and extracellular-matrix stiffness.