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

TL;DR

Deleting the P2rx7 gene worsens muscle disease in mice with α-sarcoglycan muscular dystrophy, suggesting that targeting this receptor may need careful timing and dosage.

Contribution

Genetic deletion of P2rx7 in α-sarcoglycan dystrophy mice unexpectedly worsens disease severity compared to pharmacological inhibition.

Findings

Genetic deletion of P2rx7 in Sgca–/– mice increased fibrosis and immune cell infiltration in diaphragm tissue.

P2X4R was co-expressed with immune cells in dystrophic muscle, suggesting a compensatory role.

Pharmacological P2X7R inhibition may have different effects than genetic deletion in dystrophic muscle.

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.

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