Glucocorticoids Improve Myogenic Differentiation In Vitro by Suppressing the Synthesis of Versican, a Transitional Matrix Protein Overexpressed in Dystrophic Skeletal Muscles

Abstract

In Duchenne muscular dystrophy (DMD), a dysregulated extracellular matrix (ECM) directly exacerbates pathology. Glucocorticoids are beneficial therapeutics in DMD, and have pleiotropic effects on the composition and processing of ECM proteins in other biological contexts. The synthesis and remodelling of a transitional versican-rich matrix is necessary for myogenesis; whether glucocorticoids modulate this transitional matrix is not known. Here, versican expression and processing were examined in hindlimb and diaphragm muscles from mdx dystrophin-deficient mice and C57BL/10 wild type mice. V0/V1 versican (Vcan) mRNA transcripts and protein levels were upregulated in dystrophic compared to wild type muscles, especially in the more severely affected mdx diaphragm. Processed versican (versikine) was detected in wild type and dystrophic muscles, and immunoreactivity was highly associated with newly regenerated myofibres. Glucocorticoids enhanced C2C12 myoblast fusion by modulating the expression of genes regulating transitional matrix synthesis and processing. Specifically, Tgfβ1, Vcan and hyaluronan synthase-2 (Has2) mRNA transcripts were decreased by 50% and Adamts1 mRNA transcripts were increased three-fold by glucocorticoid treatment. The addition of exogenous versican impaired myoblast fusion, whilst glucocorticoids alleviated this inhibition in fusion. In dystrophic mdx muscles, versican upregulation correlated with pathology. We propose that versican is a novel and relevant target gene in DMD, given its suppression by glucocorticoids and that in excess it impairs myoblast fusion, a process key for muscle regeneration.

Treatment of Dystrophic mdx Mice with an ADAMTS-5 Specific Monoclonal Antibody Increases the Ex Vivo Strength of Isolated Fast Twitch Hindlimb Muscles

“…Here we report that ADAMTS-5 is highly expressed in dystrophic hindlimb muscles from mdx mice and upregulated in regions of regeneration and inflammation. The co-localization of ADAMTS-5 with desmin positive muscle cells in dystrophic hindlimb muscles aligns with previous observations by our laboratory that versikine, the bioactive product of versican remodeling by ADAMTS versicanases, is also co-localized with newly regenerated desmin positive muscle fibers and muscle progenitor cells in dystrophic muscles [17]. The immunoreactivity of ADAMTS-5 in regions of mononuclear infiltrate and inflammation are also consistent with the co-localization of versikine and infiltrating macrophages in dystrophic muscle [10].…”

“…Unexpectedly, versikine was readily detected in EDL muscle cross-sections from both ADAMTS-5 mAb and IgG treated mdx mice. In concordance with previously published findings, immunoreactivity was localized to the endomysium and to myonuclei ( Figure 4A,B) [17]. When quantitatively assessed using immunoblotting, versikine protein content in either EDL or soleus muscles did not significantly differ between mdx mice treated with the ADAMTS-5 mAb or the IgG control ( Figure 4C,D).…”

“…Proteins were then transferred to PVDF membranes using a Turbo Blot Transfer System (Bio-Rad) at 2.5 A and 25 V for 12 min. Immunoblotting for versikine (anti-DPEAAE neo-epitope; Thermo Fisher Scientific, PA1-1748A) was performed as previously described [17]. Blots were imaged using ECL chemiluminescence.…”

“…Fibrosis in dystrophic muscles from patients with DMD and mdx mice (the murine model of DMD) is characterized by the upregulation of mature and provisional matrix proteins and proteases, including ADAMTS-5, V0/V1 versican, and the catalytically processed versikine fragment [10,[16][17][18][19][20]. This chronic pro-fibrotic state leads to aberrant growth factor and cytokine signaling (including TGFβ), excess inflammation, failed myogenesis, and further matrix expansion.…”

“…Interestingly, ADAMTS-1 released by macrophages following injury stimulates satellite cell activation [31], perhaps through versican remodeling in the satellite cell niche [25]. Versican remodeling by ADAMTS versicanases has been reported in dystrophic muscles from mdx mice and patients with DMD, as indicated by the co-localization of versikine to regions of regeneration and inflammation [10,17]. Adamts-5 mRNA transcripts are upregulated in hindlimb muscles from mdx mice [19].…”