Congenital muscular dystrophies (CMDs) are progressive debilitating diseases without cures. Many CMDs disrupt the adhesion of muscle cells to their surrounding extracellular matrix (ECM). Muscle-ECM adhesion is critical for muscle development, homeostasis, regeneration, and resilience to stress. Mutations in genes that modulate muscle-ECM adhesion frequently lead to CMDs. For example, Dystroglycan (DG) and Integrin alpha7 (Itga7) are transmembrane ECM receptors that, when mutated, result in CMDs. Whether and/or how these transmembrane receptors interact during muscle development/homeostasis is not known. In addition, the roles that post-translational modification of DG plays in modulating both the ECM proper and muscle-ECM adhesion are not known. We previously found that exogenous NAD+ potentiates ECM deposition and that NAD+ improves dystrophic phenotypes in zebrafish lacking either DG or Itga7. The basic cell biological mechanisms that underlie NAD+-mediated improvement in muscle-ECM adhesion are not well understood. Our long-term goal is to understand how signaling between muscle cells and their ECM mediates muscle health. Secondary Dystroglycanopathies are a subset of CMDs that result from mutations in genes that are necessary for glycosylation of DG, which is necessary for muscle-ECM adhesion. GDP-mannose, synthesized by GMPPB, is essential for glycosylation reactions. Mutations in GMPPB result in GMPPB-associated Dystroglycanopathy. Preliminary data show that muscle development, homeostasis, and regeneration are disrupted in gmppb mutants. In contrast to our previous data showing NAD+ improves ECM deposition in dg- deficient zebrafish, preliminary data show that NAD+ does not improve muscle structure in gmppb mutants. In this grant, we will compare and contrast the mechanisms underlying the effects of DG glycosylation and NAD+ on muscle development, homeostasis, and regeneration. Our central hypothesis is that both NAD+ and gmppb regulate muscle cell adhesion by altering sarcolemma architecture and ECM organization.
In Aim 1 we will test the hypothesis that NAD+ increases cell adhesion in DG mutant zebrafish by increasing Itga7 clustering; and that hypoglycosylated DG disrupts sarcolemma architecture and prevents NAD+-mediated Itga7 clustering and increased cell adhesion. We will do this with a combination of longitudinal light sheet microscopy studies and super-resolution microscopy.
In Aim 2 we will identify new muscle cell adhesion regulators through comparative studies of dysregulated muscle development in three zebrafish models of muscular dystrophy. We will take an unbiased approach to identify ECM regulatory nodes by using network modeling and network resilience analysis of co-expressed coding and non-coding genes. Completion of this grant will provide new insight into how cell-ECM adhesion mediates muscle development and homeostasis in vertebrate models of CMDs. These basic in vivo cell biological studies are crucial to provide a foundational understanding of the interplay between transmembrane receptors, ECM regulation, and cell adhesion.
We use the zebrafish as a genetically and embryologically accessible vertebrate model of congenital muscular dystrophies, in particular as a model of dystroglycanopathies. Adhesion of muscle cells to their surrounding extracellular matrix is disrupted in dystroglycanopathies. In this grant, we propose to focus on how NAD+ and GMPPB modulate organization of the muscle cell membrane and adhesion to the extracellular matrix.
