Defective myoblasts identified in Duchenne muscular dystrophy.

Abstract

A defect in the proliferative capacity of satellite cells, mononucleated precursors of mature muscle fibers, was found in clonal analyses of cells cultured from Duchenne muscular dystrophy (DMD) patients. The total yield of myoblasts per gram of muscle biopsy was decreased to 5% of normal. Of the DMD myoblast clones obtained, a large proportion -contained a morphological class of flat distended cells that had an increased generation time and ceased to proliferate beyond 100-1,000 cells but could be induced to fuse and form myotubes. The altered muscle phenotype was detected in all cultures from DMD patients but was rarely found among myoblasts of controls. By age 14 yr, it comprised as many as 90% of DMD myoblasts. The remaining DMD myoblast clones, which initially grew well, had severely impaired proliferative capacity upon passage and further cultivation. Eventually all myoblasts from DMD muscle tissue exhibited defective growth potential. In contrast, the fibroblast yield and proliferative capacity from DMD samples did not differ from normal. Based on these findings, we propose a hypothesis for the etiology of DMD: Dividing myoblasts are required for muscle growth and maintenance, and the limited capacity of DMD myoblasts to grow is directly related to the progressive muscle degeneration characteristic of the disease.Duchenne muscular dystrophy (DMD) is a chronic, lethal Xlinked disease that affects 1 in 4,800 males (1). Although DMD is well defined clinically, the cellular and biochemical basis for the disease is unknown. A major problem in the biological study of muscular dystrophy has been the heterogeneous nature of muscle tissue. Muscle is composed of a mixture of cell types present in variable ratios, especially in disease states. When muscle is dissociated and the cells plated in culture, both myoblasts and fibroblasts are obtained and the rate of proliferation of the fibroblasts frequently exceeds that of the myoblasts. DMD muscle cultures have been compared with normal muscle cultures and a number of differences reported, including increased collagen synthesis (2), decreased specific activity of creatine kinase (3), increased ratio of nonmuscle to muscle creatine kinase isozymes (3-5), altered lipid biosynthesis (6), and clustered myotube morphology (5,7,8). However, it is unclear whether these differences are intrinsic to the DMD myoblasts and related to the disease or a reflection of the proportion of fibroblasts present in the mixed cultures analyzed.We have developed methods for obtaining pure populations of human myoblasts that can be studied under standardized conditions (9). Our methods use postnatal muscle as a source.Consequently, the myoblasts obtained in culture are muscle satellite cells, the mononucleated myoblasts of mature muscle fibers, positioned between the basement membrane and the sarcolemma (10). It is these satellite cells that are capable of proliferating in vitro (11, 12) and are probably responsible for postnatal muscle regeneration and fiber growth in vivo. We have...

Normal growth and regenerating ability of myoblasts from unaffected muscles of facioscapulohumeral muscular dystrophy patients

“…In fact, the proportion of CD56-and desmin-positive FSHD myoblasts remained high throughout the production, while DMD myoblasts have been reported to display an inexorable and rapid decline during consecutive passages [11][12][13]51 (personal communication), and to undergo premature senescence, although they generally keep the ability to form myotubes in vitro. FSHD myoblasts Immunodeficient Rag mice (12 weeks old) were injected with patient or control myoblasts at 1-week interval (n ¼ 2-5 muscles for each condition).…”

“…As a consequence, the remaining cells cannot sustain the large-scale expansions required for perpetual muscle regeneration, or use in clinical practice. [11][12][13] For this reason, the DMD myoblasts have been prepared from healthy donors, and MT has been performed in a heterologous context. The MT concept has been validated in normal and dystrophic mouse models, where injection of healthy myoblasts increased muscle mass and function.…”

Ex vivo gene transfer using adenovirus-mediated full-length dystrophin delivery to dystrophic muscles

“…Since muscle weakness in DMD patients is not typically noticed until approximately 5 years of age, it appears that the dystrophic muscle of younger children is functionally rescued by the large population of satellite cells that continuously regenerate the chronically damaged myofibers. As the patient ages and the satellite cells eventually undergo mitotic senescence, 68,69 the progressive muscle weakness associated with this disease becomes manifest. The ex vivo approach permits not only the restoration of myoblasts capable of regenerating myofibers and potentially reconstructing the dystrophic muscle, but also permits the introduction of normal cDNA copies of dystrophin to the dystrophic mdx muscle.…”

MicroRNA-199a is induced in dystrophic muscle and affects WNT signaling, cell proliferation, and myogenic differentiation

“…2,3 Dystrophic muscle is known to undergo cycles of regeneration to form new myofibers as a consequence of myofibers loss and myoblast cell dysregulation. 4 WNT (wingless-type MMTV integration site family) signaling factors are known to regulate both the maintenance of muscle satellite cells (MSCs), and their ability to differentiate into myofibers. [5][6][7] Conversely, both dystrophic and aged skeletal muscle display activated WNT signaling resulting in increased muscle fibrosis.…”