Genetic Background Affects Properties of Satellite Cells and mdx Phenotypes

Duchenne muscular dystrophy (DMD) is the most common lethal genetic disorder of children. The mdx (C57BL/10 background, C57BL/10-mdx) mouse is a widely used model of DMD, but the histopathological hallmarks of DMD, such as the smaller number of myofibers, accumulation of fat and fibrosis, and insufficient regeneration of myofibers, are not observed in adult C57BL/10-mdx except for in the diaphragm. In this study, we showed that DBA/2 mice exhibited decreased muscle weight, as well as lower myofiber numbers after repeated degeneration–regeneration cycles. Furthermore, the self-renewal efficiency of satellite cells of DBA/2 is lower than that of C57BL/6. Therefore, we produced a DBA/2-mdx strain by crossing DBA/2 and C57BL/10-mdx. The hind limb muscles of DBA/2-mdx mice exhibited lower muscle weight, fewer myofibers, and increased fat and fibrosis, in comparison with C57BL/10-mdx. Moreover, remarkable muscle weakness was observed in DBA/2-mdx. These results indicate that the DBA/2-mdx mouse is a more suitable model for DMD studies, and the efficient satellite cell self-renewal ability of C57BL/10-mdx might explain the difference in pathologies between humans and mice.Duchenne muscular dystrophy (DMD) is a progressive and lethal X-linked muscular disorder caused by mutations in the dystrophin gene.¹ The dystrophin gene encodes a 427-kDa cytoskeletal protein that forms the dystrophin/glycoprotein complex at the sarcolemma with α- and β-dystroglycans, α-, β-, γ-, and δ-sarcoglycans, and other molecules, and links the cytoskeleton of myofibers to the extracellular matrix in skeletal muscle.^2,3 The lack of dystrophin in the sarcolemma disturbs the assembly of the dystrophin/glycoprotein complex and causes instability of the muscle membrane, leading to muscle degeneration and myofiber loss. The histopathological hallmarks of DMD include degeneration, necrosis, accumulation of fat and fibrosis, and insufficient regeneration of myofibers accompanied by a loss of myofibers.⁴ Therefore, the manifestations of DMD are considered to result from an imbalance between degeneration and regeneration.The function and structure of dystrophin has been elucidated by studies of a variety of dystrophin-deficient animals. Among these animal models, the mdx mouse (the correct nomenclature is C57BL/10-Dmd^mdx), first described in1984, is the most prolific. A spontaneous mutation (mdx) arose in an inbred colony of C57BL/10 mice, which have a high level of serum pyruvate kinase.⁵ The muscle pathology of the mice includes active fiber necrosis, cellular infiltration, a wide range of fiber sizes, and numerous centrally nucleated regenerating fibers. However, in contrast to DMD, replacement of muscle with fat and fibrosis is not prominent, and no losses of muscle fiber and muscle weight are observed in the skeletal muscle of mdx mice except in the diaphragm.^6,7 In contrast, most of the limb muscles of the mdx mouse maintain hypertrophy and increased skeletal muscle mass throughout much of their life span.⁸ One reason for the difference between DMD and mdx is explained by the up-regulation of expression of utrophin, a homolog of dystrophin.^9,10 Another reason has been supposed to be the excellent regeneration capacity of mdx compared with DMD. However, this hypothesis has not been verified.Regeneration of skeletal muscle depends on the competence of muscle satellite cells. Muscle satellite cells, which account for 2 to 5% of the total nuclei in adult skeletal muscle, play a major role in muscle regeneration.¹¹ Under normal conditions, satellite cells are found external to the myofiber plasma membrane and beneath the muscle basal lamina,¹² and they are mitotically quiescent in adult skeletal muscle.¹³ When activated by muscle damage, satellite cells proliferate, differentiate, fuse with each other or injured myofibers, and eventually regenerate mature myofibers. During the regenerative processes, satellite cells not only produce large amounts of muscle, but also renew themselves to maintain their own population.¹⁴ In fact, it is reported that the satellite cell pool of C57BL/10 continues to respond efficiently even when the skeletal muscle is subjected to as many as 50 cycles of severe damage.¹⁵ Therefore, it is thought that maintenance of the satellite cell pool is indispensable to retain the long-term regenerative potential for skeletal muscle injury, including in muscular dystrophies.To investigate genetic differences in long-term regeneration potential, we first induced repeated degeneration–regeneration cycles in four inbred strains of mice. Among these strains, C57BL/6, a widely used strain akin to C57BL/10, was tolerant of repeated injury. This is consistent with the results of C57BL/10 previously described.¹⁵ In contrast, among four inbred strains, DBA/2 mice exhibited the most remarkable skeletal muscle loss and impaired regeneration after repeated injury. Importantly, the self-renewal potential of DBA/2 satellite cells was significantly lower than that of C57BL/6. In addition, in vitro colony formation and proliferation assays indicated that intrinsic difference between C57BL/6 and DBA/2 satellite cells exist. Finally, we crossed the mdx genotype with the DBA/2 for more than five generations. At the fifth backcross, the mice are not yet fully congenic (D2.B10-DMD^mdx), and thus we refer to them as DBA/2-mdx hereafter. We investigated their phenotypes. Intriguingly, severe loss of skeletal muscle weight, decreased myofiber number, increased fat and fibrosis volume, and apparent muscle weakness were observed in the DBA/2-mdx mice. These results indicate that the intrinsic genetic program affects the properties of satellite cells, and DBA/2-mdx will be a more useful model of DMD than C57BL/10-mdx. It is also speculated that the high self-renewal potential of C57BL/10 satellite cells might explain the difference in pathologies between humans and mice.