Dottorato in MORFOGENESI E INGEGNERIA TISSUTALE

Titolo della tesi: “𝛃 -sarcoglycanopathy (LGMDR4): a study of muscle diversity using a Sgc𝛃 KO mouse model.”

Limb Girdle Muscular Dystrophy type R4 (LGMD R4 or β- sarcoglycanopathy) is characterized by severe muscle alterations, resulting in poor prognosis in few years. Despite a common genetic mutation, some muscles resist to degeneration and even develop hypertrophy, as it happens in human calf or tongue muscles. This study uses a Sgcb null mouse model to investigate why different muscles degenerates with different kinetics and features. The histology and transcriptome of nine different skeletal muscles and the heart were evaluated. Histological analysis was performed on mice at 1, 6 and 12 months of age, corresponding to 3 different stages of disease progression. Proximal muscles such as pectoralis, quadriceps and lumbaris were the most altered by the mutation, in terms of muscle degeneration, scar deposition and muscle fiber size and metabolism, while distal muscles such as extensor digitorum longus (EDL), gastrocnemius and soleus were less affected. Transcriptomic analysis of the selected muscles revealed significant differences in gene expression profiles between WT and KO mice. In physiological conditions, we observed a clear clustering of muscle tissues, with cardiac muscle,soleus and diaphragm presenting the major profile differences compared to the rest of skeletal muscles. In Sgcβ KO mice, we observed greater transcriptional variability and dispersed clustering, indicating increased heterogeneity across muscles. Despite this, soleus and diaphragm remain separated from other skeletal muscles, suggesting their distinct metabolic characteristics and possible different responses to beta-sarcoglycan deficiency. Thirteen differentially expressed genes were consistently identified as potential markers of the pathology across all KO muscles analyzed. Further analysis of differentially expressed genes between WT and KO mice showed an enrichment in biological processes related to immunity, while processes associated with muscle contraction and energy metabolism were downregulated. Furthermore, for the first time, we showed that the entire sarcoglycan complex is enriched at the neuromuscular junction (NMJ). Morphological alterations in both pre- and post-synaptic membranes of the NMJ, and decreased membrane potential were observed in the absence of beta- sarcoglycan gene. In addition, we demonstrated that the localization of sarcoglycans depends on the nerve presence. These new findings suggest that sarcoglycans function at the NMJ could contribute to heterogeneity among muscles in beta sarcoglycanopathy. In conclusion, this exploratory study highlighted that murine beta- sarcoglycanopathy is characterized histologically by a proximal- distal gradient of muscle involvement. Moreover, the enrichment of the sarcoglycan complex at the NMJ and its dependency on nerve signals revealed a new potential role for sarcoglycans beyond their canonical function as part of the DGC complex.