A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90

P2RX7 is an ATP-gated ion channel, which can also exhibit an open state with a considerably wider permeation. However, the functional significance of the movement of molecules through the large pore (LP) and the intracellular signaling events involved are not known. Here, analyzing the consequences of P2RX7 activation in primary myoblasts and myotubes from the <i>Dmd^mdx</i> mouse model of Duchenne muscular dystrophy, we found ATP-induced P2RX7-dependent autophagic flux, leading to CASP3-CASP7-independent cell death. P2RX7-evoked autophagy was triggered by LP formation but not Ca²⁺ influx or MAPK1-MAPK3 phosphorylation, 2 canonical P2RX7-evoked signals. Phosphoproteomics, protein expression inference and signaling pathway prediction analysis of P2RX7 signaling mediators pointed to HSPA2 and HSP90 proteins. Indeed, specific HSP90 inhibitors prevented LP formation, LC3-II accumulation, and cell death in myoblasts and myotubes but not in macrophages. Pharmacological blockade or genetic ablation of <i>p2rx7</i> also proved protective against ATP-induced death of muscle cells, as did inhibition of autophagy with 3-MA. The functional significance of the P2RX7 LP is one of the great unknowns of purinergic signaling. Our data demonstrate a novel outcome—autophagy—and show that molecules entering through the LP can be targeted to phagophores. Moreover, we show that in muscles but not in macrophages, autophagy is needed for the formation of this LP. Given that P2RX7-dependent LP and HSP90 are critically interacting in the ATP-evoked autophagic death of dystrophic muscles, treatments targeting this axis could be of therapeutic benefit in this debilitating and incurable form of muscular dystrophy.

P2RX7是一种ATP门控离子通道，还可呈现通透性显著更宽的开放状态。然而，分子经大孔隙（large pore, LP）转运的功能意义，以及所涉及的细胞内信号事件，目前仍未明确。本研究针对杜氏肌营养不良症（Duchenne muscular dystrophy, DMD）的*Dmd^mdx*小鼠模型的原代成肌细胞与肌管，分析P2RX7激活的效应，结果发现ATP诱导的、依赖P2RX7的自噬流，最终引发不依赖半胱天冬酶3（CASP3）-半胱天冬酶7（CASP7）的细胞死亡。P2RX7诱发的自噬由大孔隙形成所触发，而非经典的P2RX7诱发信号——钙离子内流或丝裂原活化蛋白激酶1（MAPK1）-丝裂原活化蛋白激酶3（MAPK3）的磷酸化。对P2RX7信号介质的磷酸化蛋白质组学、蛋白质表达推断及信号通路预测分析，指向热休克蛋白A2（HSPA2）与热休克蛋白90（HSP90）蛋白。实验证实，特异性HSP90抑制剂可阻断成肌细胞与肌管中的大孔隙形成、LC3-II积累及细胞死亡，但对巨噬细胞无此效应。对*p2rx7*基因的药理学阻断或遗传敲除，同样可保护肌细胞免受ATP诱导的死亡，这与使用3-甲基腺嘌呤（3-MA）抑制自噬的效果一致。P2RX7大孔隙的功能意义是嘌呤能信号传导领域的重大未解之谜之一。本研究数据揭示了一种全新的结局——自噬，并证实经大孔隙进入的分子可被靶向递送至吞噬泡（phagophore）。此外，本研究表明，仅在肌肉细胞而非巨噬细胞中，自噬是大孔隙形成所必需的。鉴于在营养不良型肌肉细胞的ATP诱导自噬性死亡中，P2RX7依赖的大孔隙与HSP90存在关键的相互作用，靶向这一信号轴的治疗策略，有望为这种致残且目前无法治愈的肌营养不良症提供治疗益处。