Biology subjects

Crosbie, R. H.

Publications and source records attributed to Crosbie, R. H..

3 recordsLinked to original sources

bioRxiv2022

Myoscaffolds demonstrate differential matrix components across muscular dystrophies

Extracellular matrix (ECM) pathologic remodeling underlies many fibrotic disorders, including muscular dystrophy. Tissue decellularization removes cellular components while leaving behind ECM components. We generated "on-slide" decellularized tissue slices from genetically distinct dystrophic mouse models. The ECM of dystrophin-and sarcoglycan-deficient muscles had marked thrombospondin 4 deposition, while dysferlin-deficient muscle had excess decorin. Annexins A2 and A6 were present on all dystrophic decellularized ECMs, but annexin matrix deposition was excessive in dysferlin-deficient muscular dystrophy. Adeno-associated viral expression of annexin A6 specifically in muscle resulted in annexin A6 deposition throughout the ECM, indicating muscle as a source of this ECM protein. C2C12 myoblasts seeded onto decellularized matrices displayed differential myoblast mobility. Dystrophin-deficient decellularized matrices inhibited myoblast mobility while dysferlin-deficient decellularized matrices enhanced myoblast movement. Myoblasts treated with recombinant annexin A6 increased mobillity similar to that seen on dysferlin-deficient decellularized matrix. These findings demonstrate specific fibrotic signatures elicit effects on myoblast activity. TEASERFibrosis in muscular dystrophy has differential effects on myoblasts HIGHLIGHTSO_LISpatial architecture and composition of the ECM differ across genetically distinct forms of muscular dystrophy, especially with respect to Annexin A6 protein deposition C_LIO_LIMatrix from dystrophin-mediated muscular dystrophy inhibits myoblast movement C_LIO_LIMatrix from dysferlin-deficient muscular dystrophy promotes myoblast motility C_LIO_LIAnnexin A6 was sufficient to enhance myoblast motility C_LI

molecular biology↗

bioRxiv2022

Multi-omics analysis of sarcospan overexpression in mdx skeletal muscle reveals compensatory remodeling of cytoskeleton-matrix interactions that promote mechanotransduction pathways

BackgroundThe dystrophin-glycoprotein complex (DGC) is a critical adhesion complex of the muscle cell membrane, providing a mechanical link between the extracellular matrix (ECM) and the cortical cytoskeleton that stabilizes the sarcolemma during repeated muscle contractions. One integral component of the DGC is the transmembrane protein, sarcospan (SSPN). Overexpression of SSPN in the skeletal muscle of mdx mice (murine model of DMD) restores muscle fiber attachment to the ECM in part through an associated increase in utrophin and integrin adhesion complexes at the cell membrane, protecting the muscle from contraction-induced injury. In this study, we utilized transcriptomic and ECM protein-optimized proteomics data sets from wild-type, mdx, and mdx transgenic (mdx TG) skeletal muscle tissues to identify pathways and proteins driving the compensatory action of SSPN overexpression. MethodsThe tibialis anterior and quadriceps muscles were isolated from wild-type, mdx, and mdx TG mice and subjected to bulk RNA-Seq and global proteomics analysis using methods to enhance capture of ECM proteins. Data sets were further analyzed through the Ingenuity Pathway Analysis (QIAGEN) and integrative gene set enrichment to identify candidate networks, signaling pathways, and upstream regulators. ResultsThrough our multi-omics approach, we identified 3 classes of differentially expressed genes and proteins in mdx TG muscle, included those that were: 1) unrestored (significantly different from wild-type, but not from mdx), 2) restored (significantly different from mdx, but not from wild-type), and 3) compensatory (significantly different from both wild-type and mdx). We identified signaling pathways that may contribute to the rescue phenotype, most notably cytoskeleton and ECM organization pathways. ECM optimized-proteomics revealed an increased abundance of collagens II, V, and XI, along with {beta}-spectrin in mdx TG samples. Using Ingenuity Pathway Analysis, we identified upstream regulators that are computationally predicted to drive compensatory changes, revealing a possible mechanism of SSPN rescue through a rewiring of cell-ECM bidirectional communication. We found that SSPN overexpression results in upregulation of key signaling molecules associated with regulation of cytoskeleton organization and mechanotransduction, including Rho, RAC, and Wnt. ConclusionsOur findings indicate that SSPN overexpression rescues dystrophin deficiency partially through mechanotransduction signaling cascades mediated through components of the ECM and the cortical cytoskeleton.

molecular biology↗

bioRxiv2022

Myoscaffolds reveal laminin scarring is detrimental for stem cell function while sarcospan induces compensatory fibrosis

We developed an on-slide decellularization approach to generate acellular extracellular matrix (ECM) scaffolds that can be repopulated with various cell types to interrogate cell-ECM interactions. Using this platform, we investigated whether fibrotic ECM scarring affected human skeletal muscle progenitor cell (SMPC) functions that are essential for myoregeneration. SMPCs exhibited robust adhesion, motility, and differentiation on healthy muscle-derived myoscaffolds. All SPMC interactions with fibrotic myoscaffolds from dystrophic muscle were severely blunted including reduced motility rate and migration. Furthermore, SMPCs were unable to remodel laminin dense fibrotic scars within diseased myoscaffolds. Proteomics and structural analysis revealed that excessive collagen deposition alone is not pathological, and can be compensatory, as revealed by overexpression of sarcospan and its associated ECM receptors in dystrophic muscle. Our in vivo data also supported that ECM remodeling is important for SMPC engraftment and that fibrotic scars may represent one barrier to efficient cell therapy.

cell biology↗