Genetic Deletion of the Purinergic Receptor <i>P2rx7</i> 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 (<i>SGCA</i>) and aggravated by immune-mediated damage and fibrotic tissue replacement. Pharmacological inhibition of purinergic receptor P2X7 (P2X7R) reduced inflammation and fibrosis in <i>Sgca</i> ^-/- mice. To further define the role of P2X7R, we generated a double knockout mouse model <i>Sgca</i> ^<i>-/-</i> <i>P2rx7</i> ^{<i>‑/</i>‑}. We compared diaphragms isolated from 24-week-old <i>Sgca</i> ^-/- <i>P2rx7</i> ^+/+ and <i>Sgca</i> ^<i>-/-</i> <i>P2rx7 <i>^-/-</i> </i> mice since the diaphragmatic muscle is early and severely damaged by <i>Sgca</i> genetic loss-of-function. Unexpectedly, <i>Sgca</i> ^<i>-/-</i> <i>P2rx7^-/-</i> 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 <i>Sgca</i> ^-/- <i>P2rx7</i> ^<i>+/+</i> 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 <i>Sgca</i> ^<i>-/-</i> <i>P2rx7^-/-</i> 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 <i>P2rx7</i> 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.