Prosaposin expression in the regenerated muscles of mdx and cardiotoxin-treated mice

Abstract

The trophic factor prosaposin (PS) is strongly expressed in skeletal muscle, and reportedly, a PS-derived peptide attenuates loss of muscle mass after nerve injury in vivo and increases myoblast fusion into myotubes in vitro. However, few studies have focused on the role of PS during muscle regeneration. We examined the expression of PS in the skeletal muscles in normal, mdx, and cardiotoxin (CTX)-treated mice using immunofluorescence staining, Western blotting, and in situ hybridisation. Immunofluorescence showed intense PS immunoreactivity in the peripheral cytoplasm of uninjured myofibres of normal mice and regenerated myofibres of 8 weeks post-CTX-injection mice. In early stage CTX-treated mice (14 days and earlier), intense PS immunoreactivity was also detected in the immune cells that infiltrated damaged muscle, but it was weak for regenerating myofibres. Western blot confirmed these findings. In contrast, PS was continuously low in mdx mice in both immunofluorescence and Western blotting. In situ hybridisation confirmed the decrease of PS mRNA in regenerated myofibres and revealed the main form of PS mRNA as Pro+0 without a 9-base insertion both in normal and mdx mice. The embryonic myosin (MYH3) was clearly localized in the newly regenerated myofibres at 3, 7, and 14 days of post-CTX-injection and mdx mice, but was lower in the late stage of regenerated myofibres (28 and 56 days post-CTX injection). The inverse distribution of MYH3 and PS indicates that the PS expression is closely related to the differentiation of regenerated myofibres. Investigation of the mitogen-activated protein (MAP) kinase signal pathway showed the inversely synchronous correlation of phosphorylated ERK1/2 with myofibre PS and the synchronous correlation of phosphorylated p-38 with myofibre PS. These data suggest that PS is involved in the regulation of muscle differentiation of regenerated fibres.