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

Stearns-Reider, Kristen M; Hicks, Michael R.; Hammond, Katherine G; Reynolds, Joseph; Maity, Alok Kumar; Kurmangaliyev, Yerbol Z.; Chin, Jesse; Stieg, Adam Z.; Geisse, Nicholas A.; Hohlbauch, Spohia; Kaemmer, Stefan; Schmitt, Lauren R.; Pham, Thanh; Yamauchi, Ken; Novitch, Bennett G.; Wollman, Roy; Hansen, Kirk C.; Crosbie, Rachelle H.

doi:10.1101/2022.07.07.497559

Cited by 9 publications

(21 citation statements)

References 92 publications

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“…Thus, while Hh is sufficient to induce fibrosis, it is not necessary. As fibrotic scar tissue is a potent anti-myogenic signal 11,72,73 , this increased fibrosis could also contribute to the myogenic defects we observe after ectopic Hh activation. We also determined whether Hh de-repression could result in fibrosis.…”

Section: Discussionmentioning

confidence: 89%

Hedgehog signaling via its ligand DHH acts as cell fate determinant during skeletal muscle regeneration

Norris

Johnson

Zhou

et al. 2022

Preprint

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Failed tissue regeneration often results in fatty fibrosis, the replacement of healthy cells with fibrotic scar and fat tissue. However, the causative signals remain largely unknown. Here, we describe how the Hedgehog (Hh) signaling pathway controls the fate of fibro/adipogenic progenitors (FAPs), the cellular origin of fatty fibrosis. Using conditional mutagenesis and pharmacological Hh modulators, we identify DHH as the main ligand through which Hh restricts intramuscular fat (IMAT) formation and promotes muscle regeneration. In contrast, conditionally activating Hh specifically in FAPs skews them from an adipogenic towards a fibrogenic fate resulting in massive fibrotic scar tissue. Surprisingly, FAP-specific Hh activation also impairs myoblast differentiation leading to muscle regeneration defects. Together, our data reveal novel post-developmental functions of Hh signaling in balancing tissue regeneration and fatty fibrosis. Moreover, they provide the exciting possibility that mis-regulation of the Hh pathway with age and disease could be a major driver of pathological IMAT formation, thereby representing an attractive therapeutic target.

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Section: Discussionmentioning

confidence: 89%

Hedgehog signaling via its ligand DHH acts as cell fate determinant during skeletal muscle regeneration

Norris

Johnson

Zhou

et al. 2022

Preprint

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“…POSTN and DCN are known inhibitors of myoblast migration and differentiation. Stearns-Reider et al demonstrated that skeletal muscle progenitor cells seeded into heavily fibrotic areas of mdx dECM scaffolds had reduced migratory capacity, a rounded morphology, and reduced differentiation potential compared to mildly-fibrotic areas of mdx and WT matrices (Stearns-Reider et al, 2022).…”

Section: Discussionmentioning

confidence: 99%

“…Slides were stored at -80°C. Decellularization was performed as in Stearns-Reider et al,2022 with the following modifications (Stearns-Reider et al, 2022). Slides were allowed to thaw to room temperature for 1 hr prior to decellularization.…”

Section: Methodsmentioning

confidence: 99%

“…Additionally, genetic mutations in core ECM proteins like collagen and laminin can result in COL6-related disorders Bethlem and Ullrich muscular dystrophy and LAMA2-associated congenital muscular dystrophies respectively, illustrating the critical nature of an intact matrix (Bonnemann et al, 2014; Mendell et al, 2006). Given the role of excess matrix in contributing to muscular dystrophy pathology, we employed and optimized a recently described method of generating myoscaffolds and compared ECM in different muscular dystrophy subtypes (Stearns-Reider et al, 2022).…”

Section: Introductionmentioning

confidence: 99%

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The extracellular matrix differentially directs myoblast motility in distinct forms of muscular dystrophy

Long

Kwon

Reiser

et al. 2022

Preprint

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Pathological remodeling of the extracellular matrix (ECM) underlies many fibrotic disorders, including muscular dystrophy. In fibrotic disorders, large matricellular proteins like proteoglycans and glycoproteins, and smaller secreted factors are progressively deposited into the matrix. Tissue decellularization uses detergent treatment to remove the cellular component, leaving behind intact the matrix components. Decellularized ECMs (or dECMs) have most commonly been studied in intact organs, but more recently, this method has been adapted to tissue sections on slides. We generated dECM from genetically different mouse models of muscular dystrophies including dystrophin-deficient, gamma-sarcoglycan-deficient, and dysferlin-deficient mice, as well as analyzing distinct background mouse strains known to alter the ECM. Formalin fixed samples were assessed for complete decellularization, preservation of ECM architecture, and protein retention using histological staining and immunofluorescence microscopy. Excess collagen deposition correlated with fibrosis severity across the different dystrophic models. Each muscular dystrophy subtype demonstrated excess decorin deposition in the matrix. Dystrophin- and gamma-sarcoglycan-deficient muscles displayed excess thrombospondin 4, while dysferlin-deficient muscle had excess decorin but not excess thrombospondin 4. Annexins A2 and A6 were increased across all dystrophic dECMs, but annexin deposition was greatest in dysferlin-deficient muscular dystrophy. In this model, annexin A2 was diffusely increased but annexin A6 was found in discrete regions of marked excess protein within the matrix. These studies highlight the differential nature of the core matrisome and its associated proteins in different forms of muscular dystrophy, where these proteins are positioned to influence the surrounding myofibers and inflammatory infiltrate.

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“…The mass spectrometry dataset was first published in part in Stearns-Reider et al (47). Quadriceps muscles were harvested from 20-week-old male wild-type, mdx , and mdx TG (line 3) mice and snap frozen in liquid nitrogen.…”

Section: Methodsmentioning

confidence: 99%

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

McCourt

Stearns-Reider

Mamsa

et al. 2022

Preprint

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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 (mdx TG) 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 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. Results: Through 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 β-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. 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.

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

Cited by 9 publications

References 92 publications

Hedgehog signaling via its ligand DHH acts as cell fate determinant during skeletal muscle regeneration

Hedgehog signaling via its ligand DHH acts as cell fate determinant during skeletal muscle regeneration

The extracellular matrix differentially directs myoblast motility in distinct forms of muscular dystrophy

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

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