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Inhibition of Postn Rescues Myogenesis Defects in Myotonic Dystrophy Type 1 Myoblast Model.

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.

SUBMITTER: Shen X

PROVIDER: S-EPMC8417118 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Myotonic dystrophy type 1 (DM1) is the most common type of adult muscular dystrophy caused by the expanded triple-nucleotides (CUG) repeats. Myoblast in DM1 displayed many defects, including defective myoblast differentiation, ribonuclear foci, and aberrant alternative splicing. Despite many were revealed to function in DM1, microRNAs that regulated DM1 via directly targeting the expanded CUG repeats were rarely reported. Here we discovered that miR-322/-503 rescued myoblast defects in DM1 cell model by targeting the expanded CUG repeats. First, we studied the function of miR-322/-503 in normal C2C12 myoblast cells. Downregulation of miR-322/-503 significantly hindered the myoblast differentiation, while miR-322/-503 overexpression promoted the process. Next, we examined the role of miR-322/-503 in the DM1 C2C12 cell model. miR-322/-503 was downregulated in the differentiation of DM1 C2C12 cells. When we introduced ectopic miR-322/-503 expression into DM1 C2C12 cells, myoblast defects were almost fully rescued, marked by significant improvements of myoblast differentiation and repressions of ribonuclear foci formation and aberrant alternative splicing. Then we investigated the downstream mechanism of miR-322/-503 in DM1. Agreeing with our previous work, Celf1 was proven to be miR-322/-503's target. Celf1 knockdown partially reproduced miR-322/-503's function in rescuing DM1 C2C12 differentiation but was unable to repress ribonuclear foci, suggesting other targets of miR-322/-503 existed in the DM1 C2C12 cells. As the seed regions of miR-322 and miR-503 were complementary to the CUG repeats, we hypothesized that the CUG repeats were the target of miR-322/-503. Through expression tests, reporter assays, and colocalization staining, miR-322/-503 was proved to directly and specifically target the expanded CUG repeats in the DM1 cell model rather than the shorter ones in normal cells. Those results implied a potential therapeutic function of miR-322/-503 on DM1, which needed further investigations in the future.

Project description:Myotonic dystrophy type 1 (DM1) and 2 (DM2) are autosomal dominant degenerative neuromuscular disorders characterized by progressive skeletal muscle weakness, atrophy, and myotonia with progeroid features. Although both DM1 and DM2 are characterized by skeletal muscle dysfunction and also share other clinical features, the diseases differ in the muscle groups that are affected. In DM1, distal muscles are mainly affected, whereas in DM2 problems are mostly found in proximal muscles. In addition, manifestation in DM1 is generally more severe, with possible congenital or childhood-onset of disease and prominent CNS involvement. DM1 and DM2 are caused by expansion of (CTG•CAG)n and (CCTG•CAGG)n repeats in the 3' non-coding region of DMPK and in intron 1 of CNBP, respectively, and in overlapping antisense genes. This critical review will focus on the pleiotropic problems that occur during development, growth, regeneration, and aging of skeletal muscle in patients who inherited these expansions. The current best-accepted idea is that most muscle symptoms can be explained by pathomechanistic effects of repeat expansion on RNA-mediated pathways. However, aberrations in DNA replication and transcription of the DM loci or in protein translation and proteome homeostasis could also affect the control of proliferation and differentiation of muscle progenitor cells or the maintenance and physiological integrity of muscle fibers during a patient's lifetime. Here, we will discuss these molecular and cellular processes and summarize current knowledge about the role of embryonic and adult muscle-resident stem cells in growth, homeostasis, regeneration, and premature aging of healthy and diseased muscle tissue. Of particular interest is that also progenitor cells from extramuscular sources, such as pericytes and mesoangioblasts, can participate in myogenic differentiation. We will examine the potential of all these types of cells in the application of regenerative medicine for muscular dystrophies and evaluate new possibilities for their use in future therapy of DM.

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Inhibition of Postn Rescues Myogenesis Defects in Myotonic Dystrophy Type 1 Myoblast Model.

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