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

Abstract

Background The 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 (mdxTG) skeletal muscle tissues to identify pathways and proteins driving the compensatory action of SSPN overexpression. Methods The tibialis anterior and quadriceps muscles were isolated from wild-type, mdx, and mdxTG 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. Results Through our multi-omics approach, we identified 3 classes of differentially expressed genes and proteins in mdxTG muscle, including 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 β-spectrin in mdxTG 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 Yap1, Sox9, Rho, RAC, and Wnt. Conclusions Our findings indicate that SSPN overexpression rescues dystrophin deficiency partially through mechanotransduction signaling cascades mediated through components of the ECM and the cortical cytoskeleton.

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

“…Taken together, the changes observed in the mdx TG ECM suggest that upregulation of these developmental and cartilaginous collagens may be beneficial for muscle regeneration. This compensatory ECM phenotype additionally reveals targets that could be leveraged to develop improved therapeutic strategies to address muscle pathology in several disease contexts 81 .…”

Uncovering the Gene Regulatory Network of Endothelial Cells in Mouse Duchenne Muscular Dystrophy: Insights from Single-Nuclei RNA Sequencing Analysis

“…This network acts as a barrier to restrict adhesion molecules and a filter for invasive protrusions, helping to create the conditions for successful membrane fusion [ 31 ]. Interestingly, McCount JL et al [ 32 ] found that sarcospan overexpression in DMD mut skeletal muscle can rescues dystrophin deficiency partially through mechanotransduction signaling cascades mediated through a rewiring of cell-extracellular matrix (ECM) bidirectional communication, along with spectrin beta by multi-omics analysis.…”

How Can Proteomics Help to Elucidate the Pathophysiological Crosstalk in Muscular Dystrophy and Associated Multi-System Dysfunction?

“…Multi-omics approaches have a great potential to improve our understanding of complex human disease mechanisms [ 473 ] and establish systems biological concepts [ 474 ], including the systems biology of skeletal muscles [ 475 ]. The application of multi-omics has already been used to study crucial aspects of skeletal muscle cell biology in health and disease [ 476 , 477 , 478 , 479 ] and been applied to certain aspects of the field of dystrophinopathy research, including the integrative screening of dystrophic animal models [ 277 , 480 , 481 , 482 ], the evaluation of immune responses in muscular dystrophy [ 483 ], myogenic remodeling by human pluripotent stem cells [ 484 ], astrocyte-related abnormalities [ 485 ] and dystrophinopathy-associated cardiomyopathy [ 324 , 331 ]. The main techniques used for proteomics-centric and multi-omics studies have been recently reviewed by Rajczewski et al [ 486 ].…”