Duchenne muscular dystrophy (DMD) is an X-linked lethal muscle disorder caused by mutations in the Dmd gene encoding Dystrophin12. DMD model animals, such as mdx mice and canine X-linked muscular dystrophy dogs, have been widely utilized in the development of a treatment for DMD3. Here, we demonstrate the generation of Dmd-mutated rats using a clustered interspaced short palindromic repeats (CRISPR)/Cas system, an RNA-based genome engineering technique that is also adaptive to rats. We simultaneously targeted two exons in the rat Dmd gene, which resulted in the absence of Dystrophin expression in the F0 generation. Dmd-mutated rats exhibited a decline in muscle strength, and the emergence of degenerative/regenerative phenotypes in the skeletal muscle, heart, and diaphragm. These mutations were heritable by the next generation, and F1 male rats exhibited similar phenotypes in their skeletal muscles. These model rats should prove to be useful for developing therapeutic methods to treat DMD.
Duchenne muscular dystrophy (DMD) is a progressive disease characterised by chronic muscle degeneration and inflammation. Our previously established DMD model rats (DMD rats) have a more severe disease phenotype than the broadly used mouse model. We aimed to investigate the role of senescence in DMD using DMD rats and patients. Senescence was induced in satellite cells and mesenchymal progenitor cells, owing to the increased expression of CDKN2A, p16- and p19-encoding gene. Genetic ablation of p16 in DMD rats dramatically restored body weight and muscle strength. Histological analysis showed a reduction of fibrotic and adipose tissues invading skeletal muscle, with increased muscle regeneration. Senolytic drug ABT263 prevented loss of body weight and muscle strength, and increased muscle regeneration in rats even at 8 months—the late stage of DMD. Moreover, senescence markers were highly expressed in the skeletal muscle of DMD patients. In situ hybridization of CDKN2A confirmed the expression of it in satellite cells and mesenchymal progenitor cells in patients with DMD. Collectively, these data provide new insights into the integral role of senescence in DMD progression.
Sarcopenia is the age-related loss of skeletal muscle mass and function. Skeletal muscle comprises diverse progenitor cells, including mesenchymal progenitor cells (MPCs), which normally support myogenic cell function but cause a decline in skeletal muscle function after differentiating into fibrous/adipose tissue. Cellular senescence is a form of persistent cell cycle arrest caused by cellular stress, including oxidative stress, and is accompanied by the acquisition of senescence-associated secretory phenotype (SASP). Here, we found γH2AX+ senescent cells appeared in the interstitium in skeletal muscle, corresponding in position to that of MPCs. H2O2 mediated oxidative stress in 2G11 cells, a rat MPC clone previously established in our laboratory, successfully induced senescence, as shown by the upregulation of p21 and SASP factors, including IL-6. The senescent 2G11 cells lost their fibro/adipogenic potential, but, intriguingly, coculture of myoblasts with senescent 2G11 cells abrogated the myotube formation, which coincided with the downregulation of myomaker, a muscle-specific protein involved in myogenic cell fusion; however, forced expression of myomaker could not rescue this abrogation. These results suggest that senescent MPCs in aged rat skeletal muscle lose their fibro/adipogenic potential, but differ completely from undifferentiated progenitor cells in that senescent MPCs suppress myoblast fusion and thereby potentially accelerate sarcopenia.
Dystrophin, encoded by the DMD gene on the X chromosome, stabilizes the sarcolemma by linking the actin cytoskeleton with the dystrophin-glycoprotein complex (DGC). In-frame mutations in DMD cause a milder form of X-linked muscular dystrophy, called Becker muscular dystrophy (BMD), characterized by the reduced expression of truncated dystrophin. So far, no animal model with in-frame mutations in Dmd has been established. As a result, the effect of in-frame mutations on the dystrophin expression profile and disease progression of BMD remains unclear. In this study, we established a novel rat model carrying in-frame Dmd gene mutations (IF rats) and evaluated the pathology. We found that IF rats exhibit reduced expression of truncated dystrophin in a proteasome-independent manner. This abnormal dystrophin expression caused dystrophic changes in muscle tissues, but did not lead to functional deficiency. We also found that the expression of additional dystrophin named dpX, which forms the DGC in the sarcolemma, is associated with the appearance of truncated dystrophin. In conclusion, the outcomes of this study contribute to the further understanding of BMD pathology and help elucidate the efficiency of dystrophin recovery treatments in Duchenne muscular dystrophy, a more severe form of X-linked muscular dystrophy.
Intramuscular adipose tissue (IMAT) formation is a hallmark of marbling in cattle. IMAT is considered to originate from skeletal muscle progenitor cells with adipogenic potential. However, the mechanism involved in IMAT formation from these progenitor cells in vivo remains unclear. In the present study, among the growth factors tested, which were known to be expressed in skeletal muscle, we found only basic fibroblast growth factor (bFGF) has a pro-adipogenic effect on skeletal muscle derived adipogenic progenitor clone, 2G11 cells. Pre-exposure of 2G11 cells to bFGF did not affect initial gene expressions of CCAAT/enhancer-binding protein (C/EBP)β and C/EBPδ, while resulting in an enhancement of subsequent expressions of C/EBPα and proliferator-activated receptor gamma (PPARγ) during adipogenesis, indicating that bFGF is acting on the transcriptional regulation of C/EBPα and PPARγ. In addition, the effect of bFGF is mediated via two types of FGF receptor (FGFR) isoforms: FGFR1 and FGFR2 IIIc, and both receptors are prerequisite for bFGF to express its pro-adipogenic effect. These results suggest that bFGF plays an important role as a key trigger of IMAT formation in vivo.
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