Sonic hedgehog therapy in a mouse model of age-associated impairment of skeletal muscle regeneration

Abstract

Sonic hedgehog (Shh) is a morphogen regulating muscle development during embryogenesis. We have shown that the Shh pathway is postnatally recapitulated after injury and during regeneration of the adult skeletal muscle and regulates angiogenesis and myogenesis after muscle injury. Here, we demonstrate that in 18-month-old mice, there is a significant impairment of the upregulation of the Shh pathway that physiologically occurs in the young skeletal muscle after injury. Such impairment is even more pronounced in 24-month-old mice. In old animals, intramuscular therapy with a plasmid encoding the human Shh gene increases the regenerative capacities of the injured muscle, in terms of Myf5-positive cells, regenerating myofibers, and fibrosis. At the molecular level, Shh treatment increases the upregulation of the prototypical growth factors, insulin-like growth factor-1 and vascular endothelial growth factor. These data demonstrate that Shh increases regeneration after injury in the muscle of 24-month-old mice and suggest that the manipulation of the Shh pathway may be useful for the treatment of muscular diseases associated with aging.

Keywords: Aging.; Skeletal muscle regeneration; Sonic hedgehog.

Figure 1.

Impaired activation of the Sonic hedgehog (Shh) signaling pathway in injured muscles of 18- and 24-month-old mice. Shh real-time PCR was performed at Days 0, 2, 4, 7, and 14 after mechanical crush (a) and cardiotoxin (CTX) injury (b) of the tibialis anterior (TA) muscle. Real-time PCR for Gli1 was performed at the same time points for both experimental models as well (c and d). The ratio between Shh and Gli1 mRNA levels in the injured TA muscle and the contralateral TA muscle increased significantly in young mice at Days 2, 4, and 7 after injury (*p < .01). In 18- and 24-month-old mice, Shh and Gli1 upregulation after both mechanical and toxic injury was significantly lower compared with young mice (*p < .01, **p < .001). In addition, Shh upregulation was reduced in 24-month-old mice compared with 18-month-old mice at Days 2 and 4 after crush (*p < .01) and at Day 2 after CTX injury (^§ p < .05).

Figure 2.

Treatment with plasmid encoding the human Shh gene (phShh) induces functional activation of the Shh pathway in the injured tibialis anterior (TA) muscle of 18-month-old mice. Immediately after cardiotoxin (CTX) injection, phShh (40 µg) was administered to the injured TA muscle of 18-month-old mice and induced significant increment of Gli1 expression compared with empty plasmid at Day 4 after injury (*p < .01) (a). Four days after injection of 40 µg phShh, expression of human Shh is detectable in the TA muscle of one 18-month-old mouse, whereas no human Shh expression may be found in the TA muscle injected with the empty plasmid (b).

Figure 3.

Treatment with plasmid encoding the human Shh gene (phShh) increases the number of activated MSCs at the injury site. In 24-month-old mice treated with phShh (40 µg) after cardiotoxin (CTX) injury of the tibialis anterior (TA) muscle, there is a significant higher number of activated (Myf5-positive) MSCs at the site of injury (244.6 ± 65.6 vs 99.7 ± 11.2 Myf5-positive cells per section, *p < .01) (a and b). Many Myf5-positive cells (green staining) are also immunopositive for the Shh target gene Gli1 (red/yellow staining) (c).

Figure 4.

Treatment with plasmid encoding the human Shh gene (phShh) increases the number of regenerating myofibers and reduces fibrosis after cardiotoxin (CTX) injury. Representative images of fibers with centrally located nuclei (CLN) in cross-sections of 24-month-old mice treated with phShh and empty plasmid (a). The number of regenerating myofibers was significantly higher in the phShh-treated group (485.5 ± 120.7 vs 123.4 ± 72.4 fibers with CLN/mm², *p < .01) (b). Treatment with phShh also results in decreased percentage of fibrotic area per section (18.8% ± 6.1% vs 34.9% ± 2.3%, *p < .01) (c and d).

Figure 5.

Treatment with plasmid encoding the human Shh gene (phShh) increases expression of insulin-like growth factor (IGF)-1 and vascular endothelial growth factor (VEGF)165 after cardiotoxin (CTX) injury. Protein expression ratio (injured/contralateral muscle) of IGF-1 (a) and VEGF (b) is significantly increased both at Days 4 and 7 after injury in 24-month-old mice treated with phShh compared with controls (*p < .01).