Fig 2: P2RX7 activation induces LP formation and cell death in dystrophic myoblasts. (A) Examples of EtBr fluorescence in wild-type (Wt) and dystrophic Dmdmdx myoblasts following 30 min exposure to 3 mM eATP in LP buffer (left panels). Right panels show EtBr uptake following the same treatment in myoblasts isolated from Dmdmdx p2rx7-/- double-mutant mice and Dmdmdx myoblasts after inhibition of P2RX7 by 30 min preincubation with 100 µM A438079. Inset in Dmdmdx panel shows classic ‘macrophage-like’ membrane blebbing following exposure of Dmdmdx myoblasts to eATP. (B) Summary data showing EtBr uptake dependence on P2RX7 expression in Wt, Dmdmdx and Dmdmdx p2rx7-/- myoblasts exposed to 3 mM eATP for 30 min (C). eATP dose response of EtBr uptake in Wt and Dmdmdx myoblasts exposed to indicated concentrations of ATP for 30 min, with or without preincubation with 100 µM A438079. Digitonin represents permeabilized positive control. (D) PrestoBlue fluorescence (cell viability) in Wt, Dmdmdx, and Dmdmdx p2rx7-/- myoblasts following 30 min exposure to 3 mM eATP. (E) LDH release from Wt, Dmdmdx, and Dmdmdx p2rx7-/- myoblasts following 30 min exposure to 3 mM eATP with or without the P2RX7-specific inhibitor, A804598 (100 nM). Mean +/- SE, n = 5, P < 0 .05* and 0.0001***.

Fig 3: Phosphate in serum and cell conditioned media. Phosphate was measured from mouse serum and cell conditioned media using the malachite green assay. (A) Serum phosphate was higher in Dmd mdx-ßGeo P2rx7 -/- compared to Dmd mdx-ßGeo but not Dmd mdx-ßGeo P2rx7 KiKo mice (** = p <0.01, Ordinary one-way ANOVA with Holm-Šídák’s multiple comparison test). (B) Phosphate levels were higher in cell conditioned media (CM) from IMO cells than from either RAW cells or SC5 cells (p <0.001, Univariate analysis in SPSS with Tukey’s post-tests). (C) Phosphate secretion by RAW cells was unaltered by treatment with the P2x7 inhibitors AZT and Coomassie brilliant blue G (BBG). (D) Phosphate secretion by IMO cells was increased in the presence of BBG (* = p <0.05, Univariate analysis in SPSS with Tukey’s post-tests). (E) Phosphate secretion by SC5 cells was increased in the presence of AZT (* = p <0.05) and BBG (**** = p <0.0001, Univariate analysis in SPSS with Tukey’s post-tests).

Fig 4: Schematic depicting known interactions at the C terminus of P2RX7 involving ACTB, ACTN (actinin, a), MYH9 (myosin, heavy polypeptide 9, non-muscle), and heat shock proteins mediating autophagic cell death in dystrophic myoblasts following P2RX7 activation. HSPA2, HSPA8, and HSP90 have been shown to bind P2RX7 C-terminal tail, where Y550 residue plays an important role in mediating phosphorylation status of HSP90.42,50 In myoblasts, P2RX7 activation following ATP released from damaged or degenerating muscles result in activation of HSP90-mediated LP formation and autophagic flux, culminating in CASP3-CASP7 independent cell death. Inhibition of HSP90 or of autophagy prevented LP formation in myoblasts, indicating involvement of HSP90-mediated autophagy in this process. Inhibition of HSPA2 resulted in inhibition of autophagy but not LP formation or cell death.

Fig 5: P2RX7 activation induces autophagic flux in dystrophic myoblasts. (A) Representative protein gel blots of triplicate samples showing LC3-II responses in dystrophic (Dmdmdx) and wild-type (Wt) myoblasts. LC3-II formation invoked following 30 min treatment with 1 mM BzATP was blocked by the autophagy inhibitor 3-methyladenine (3-MA), while lysosomal protease inhibitors E-64d/pepstatin A and mefloquine (Mefloq.), which prevent the degradation of autophagosomes, further enhanced LC3-II accumulation following BzATP treatment. (B) Fold changes of LC3-II conversion as a marker of autophagosome formation. A significant compound increase in LC3-II was observed in cells treated with 1 mM BzATP + E-64d/pepstatin A or mefloquine indicating that specific P2RX7 activation induces the formation of autophagosomes rather than blocks their degradation. Significant decreases in LC3-II observed in cells pretreated with 3-MA may indicate that P2RX7-dependent autophagosome formation occurs via a PtdIns3K-dependent mechanism. (C) P2RX7 expression confers eATP-dependent autophagic flux response to HEK-293 cells. Note no LC3-II response in untransfected (HEK-293-P2RX7 negative, upper panel) and very significant activation in P2RX7-transfected cells (HEK-293-P2RX7 expressing, lower panel) following 30 min treatment with 1 mM BzATP. This effect was blocked by autophagy inhibitor 3-MA (5 mM) and increased by lysosomal protease inhibitors PepA and E-64d (both at 10 µg/ml). (D) Fold changes of LC3-II responses observed in BzATP-treated HEK cells from (C). Mean +/- SE, n = 3, P < 0 .05*, 0.001** and 0.0001***.