P2X7 receptor controversial role in α-Sarcoglycan Muscular Dystrophy

Limb-girdle muscular dystrophy R3 (LGMDR3), a rare genetic disorder characterized by progressive impairment of the limb, diaphragmatic and respiratory muscles, is caused by loss-of-function mutations in the α-sarcoglycan gene (SGCA) and aggravated by an immune-mediated damage. We previously demonstrated that pharmacological inhibition of the purinergic receptor P2X7 reduced inflammation and fibrosis in Sgca knock-out (KO) mice. To define the effects of P2X7 genetic ablation, we generated a double knockout mouse model Sgca-/- P2x7-/-(dKO). Sgca-/- mice expressing the wild-type form of P2X7 were used as controls (Sgca-/- P2x7+/+). Diaphragmatic muscle is early and severely damaged in LGMDR3 patients as well as in the Sgca KOs. Thus, we analyzed diaphragmatic muscle from KO and dKO mice: when compared to age-matched KO, 24 weeks old male dKO mice, displayed increased extracellular matrix deposition and augmented cellularity in fibrotic areas. Moreover, immunofluorescence stainings showed high number of CD3+ lymphocytes and IBA1+ macrophages in diaphragm from dKO mice. Notably, we observed an intense P2X4 signal, which co-localized with CD3+ and Iba1+ cells, confirming its expression by the infiltrating immune cells. Absence of rescue of the dystrophic phenotype was confirmed histologically in quadriceps of 24 weeks old dKO, although the fibrotic reaction was less enhanced than in diaphragms, suggesting a differential microenvironment influence on the receptor functions. Accordingly, flow cytometric data of limb muscle infiltrating immune cells did not reveal any difference between the two genotypes and clinically we did not observe any change in motor performances (four limb-hanging test) at 6, 12, 18 and 24 weeks of age. In conclusion, this study highlighted controversial effects of P2x7 genetic ablation in α-sarcoglycan dystrophic tissue. Future studies will focus on the molecular pathways triggered by P2X7 deletion in infiltrating immune cells. Pharmacological interventions adopting P2X7 inhibitors for dystrophic immune-mediate damage need the definition of a precise time window and dosage administration to avoid detrimental effects.