The Effect of Unacylated Ghrelin in the Skeletal Muscles of Dystrophic Mice

Background: Duchenne muscular dystrophy (DMD) is a disorder caused by mutations in the dystrophin gene, leading to progressive dysfunction and degeneration of skeletal muscle, heart, immune system, etc. When dystrophin, a scaffolding protein of the sarcolemmal dystrophin-glycoprotein complex (DGC) is mutated, the result is elevated oxidative stress, immune cell invasion (e.g., myeloid cells: monocytes, macrophages), Ca 2+ overload, damage, and fibrosis. Ghrelin is a 28-amino acid peptide hormone that circulates in acylated (AG) and unacylated (UAG) forms. While AG binds to the growth hormone secretagogue receptor (GHSR), skeletal muscles are considered void of GHSR, and UAG appears to protect against skeletal muscle wasting through an unknown mechanism. While NADPH oxidase-2 (Nox2), a primary source of oxidative stress in DMD pathology, is causal in DMD pathology, the redox mechanisms by which UAG could mitigate DMD pathology are unknown. Objective: We will test the hypothesis that UAG treatment will mitigate delivery of GHSR to skeletal muscle cells, elevation of Nox2 and redox signaling, and disruption of key scaffolding proteins in the DGC (e.g., a-syntrophin, dystrobrevin) in skeletal muscle of dystrophic mice. METHODS: Four-week-old C57BL10 (WT, mdx mice) are partitioned into three groups (n = 6/ group): (1) wild-type, (2) mdx mice, and (3) mdx mice + UAG (100µg/kg/day i.p.). After 10 days, Mice were euthanized, and the gastrocnemius muscles were dissected and frozen with O.C.T. in isopentane cooled in liquid N 2 for subsequent analysis. Tissue analysis was conducted using H&E staining, immunoblotting, and immunofluorescence. RESULTS: Dystrophic gastrocnemius muscle mass and mean fiber cross-sectional area were significantly higher than the mdx UAG group. Protein immunoblots and immunofluorescence revealed that UAG treatment significantly reduced tissue damage (IgG infiltration) was significantly greater in mdx vs. WT and was mitigated by UAG in mdx mice. H&E staining indicated improved regeneration in the mdx + UAG group vs. mdx mice. Furthermore, elevation of redox signaling proteins Nox2 and RANKL were significantly reduced by UAG administration. Importantly, GHSR was detected for the first time in dystrophic muscle, but GHSR invasion was abrogated by UAG. Further, Nrf2 was elevated in dystrophic muscle indicating improved antioxidant protection. In addition, a-syntrophin and dystrobrevin were lost in mdx mice compared with wild-types, with modest recovery of these key scaffolding proteins by UAG. CONCLUSION: Our primary novel findings include protection against upregulation of Nox2, RANKL, and GHSR in mdx muscle by unacylated ghrelin, linked to reduced damage and improved muscle fiber regeneration. In addition, there appears to be partial protection of sarcolemmal DGC proteins by UAG. Future experiments will investigate the mechanisms by which UAG reduces redox signaling in dystrophic muscle. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.