Genetic Deletion of the Purinergic Receptor P2rx7 Worsens the Phenotype of α-Sarcoglycan Muscular Dystrophy

Abstract

Limb-girdle muscular dystrophy R3 (LGMDR3), a rare genetic disorder characterized by progressive impairment of limb, diaphragmatic, and respiratory muscles, is caused by loss-of-function mutations in the α-sarcoglycan gene (SGCA) and aggravated by immune-mediated damage and fibrotic tissue replacement. Pharmacological inhibition of purinergic receptor P2X7 (P2X7R) reduced inflammation and fibrosis in Sgca^–/– mice. To further define the role of P2X7R, we generated a double knockout mouse model Sgca^–/–P2rx7^-/-. We compared diaphragms isolated from 24-week-old Sgca^–/–P2rx7^+/+ and Sgca^–/–P2rx7^–/–mice since the diaphragmatic muscle is early and severely damaged by Sgca genetic loss-of-function. Unexpectedly, Sgca^–/–P2rx7^–/– mice displayed increased extracellular matrix deposition and augmented cellularity in fibrotic areas, in particular, a higher number of CD3⁺ lymphocytes and Iba1⁺ macrophages compared to Sgca^–/–P2rx7^+/+ mice. Moreover, intense P2X4R signal colocalized with CD3⁺ and Iba1⁺ cells, confirming its expression by these infiltrating immune cells. Absence of an improvement of the dystrophic phenotype was histologically confirmed in Sgca^–/–P2rx7^–/– quadriceps, although the fibrotic reaction was milder than that in diaphragms, suggesting a differential influence of the tissue microenvironment on the receptor functions. Flow cytometric analysis of limb muscle-infiltrating immune cells revealed a decrease in NK cells. Motor performance tests did not reveal any difference between the two genotypes. In conclusion, this study identified a divergent outcome of genetic deletion of the P2rx7 gene as compared to P2X7R blockade in α-sarcoglycan dystrophic tissue, suggesting that pharmacological interventions targeting the P2X7R in dystrophic immune-mediated damage require careful definition of a precise time window and dosage.