Abstract
Myotonic dystrophy type 1 (DM1) is an inherited neuromuscular disease caused by expanded CTG repeats in the 3′ untranslated region (3′UTR) of the DMPK gene. The myogenesis process is defective in DM1, which is closely associated with progressive muscle weakness and wasting. Despite many proposed explanations for the myogenesis defects in DM1, the underlying mechanism and the involvement of the extracellular microenvironment remained unknown. Here, we constructed a DM1 myoblast cell model and reproduced the myogenesis defects. By RNA sequencing (RNA-seq), we discovered that periostin (Postn) was the most significantly upregulated gene in DM1 myogenesis compared with normal controls. This difference in Postn was confirmed by real-time quantitative PCR (RT-qPCR) and western blotting. Moreover, Postn was found to be significantly upregulated in skeletal muscle and myoblasts of DM1 patients. Next, we knocked down Postn using a short hairpin RNA (shRNA) in DM1 myoblast cells and found that the myogenesis defects in the DM1 group were successfully rescued, as evidenced by increases in the myotube area, the fusion index, and the expression of myogenesis regulatory genes. Similarly, Postn knockdown in normal myoblast cells enhanced myogenesis. As POSTN is a secreted protein, we treated the DM1 myoblast cells with a POSTN-neutralizing antibody and found that DM1 myogenesis defects were successfully rescued by POSTN neutralization. We also tested the myogenic ability of myoblasts in the skeletal muscle injury mouse model and found that Postn knockdown improved the myogenic ability of DM1 myoblasts. The activity of the TGF-β/Smad3 pathway was upregulated during DM1 myogenesis but repressed when inhibiting Postn with a Postn shRNA or a POSTN-neutralizing antibody, which suggested that the TGF-β/Smad3 pathway might mediate the function of Postn in DM1 myogenesis. These results suggest that Postn is a potential therapeutical target for the treatment of myogenesis defects in DM1.
Highlights
Myotonic dystrophy type 1 (DM1) is an autosomal inherited neuromuscular disease caused by aberrant expanded (CTG) trinucleotide repeats in the 3 untranslated region (3 UTR) of the DMPK gene
The myogenesis process is severely impaired in DM1 according to most studies (Amack et al, 2002; Timchenko et al, 2004; Kuyumcu-Martinez et al, 2007; Peng et al, 2015), several groups have reported no significant change in myogenic abilities in myoblasts derived from some DM1 patients (Jacobs et al, 1990; Loro et al, 2010)
We discovered that Postn was aberrantly upregulated during the myogenesis process of DM1 myoblast cells, from in vitro differentiation day 4, when myotubes started to form as a result of myoblast fusion
Summary
Myotonic dystrophy type 1 (DM1) is an autosomal inherited neuromuscular disease caused by aberrant expanded (CTG) trinucleotide repeats in the 3 untranslated region (3 UTR) of the DMPK gene. The expanded CTG repeats in DM1 are transcribed, along with the DMPK gene, into mRNA containing expanded CUG repeats, referred to as “toxic RNA” (Udd and Krahe, 2012) This toxic RNA forms a hairpin-like secondary structure in cell nuclei, leading to MBNL1 sequestration (Miller et al, 2000) and CELF1 upregulation (Kuyumcu-Martinez et al, 2007; Kalsotra et al, 2010). The dysregulation of MBNL1 and CELF1 leads to isoform switches of several important genes related to skeletal muscle function, including CLCN1, BIN1, TNNT2, IR, and PKM, which directly cause DM1 disease phenotypes (Philips et al, 1998; Savkur et al, 2001; Charlet et al, 2002; Mankodi et al, 2002; Ho et al, 2004; Fugier et al, 2011). The myogenesis defect is a serious problem in DM1 as it has been shown to be closely related to progressive muscle weakness and wasting (Kanadia et al, 2003; Ward et al, 2010)
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