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

Abstract

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 Dmdmdx 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 p2rx7 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.

Store-operated calcium entry contributes to abnormal Ca2+ signalling in dystrophic mdx mouse myoblasts

“…In contrast with this dogma, several studies conducted by us and others showed that dystrophic myoblasts similarly as in the case of lymphoblasts differ from wild type cells [4,5,34,45]. For example, mdx myoblasts have altered expression and function of P2X7 receptors, leading to abnormal Ca 2+ influx, ERK phosphorylation [4,5] and triggering a unique mechanism of autophagic cell death [6]. Moreover, dystrophin knockdown in adult muscles did not produce any pathology indicating that early dystrophin expression is crucial [46].…”

“…Exposure of the dystrophic (mdx) mouse myoblasts to extracellular ATP triggers a significant increase in [Ca 2+ ] c , with both P2X and P2Y receptors involved. Probing the P2X (ionotropic) arm of this abnormal purinergic response we identified P2RX7 as the main receptor responsible [4,5] and also discovered a novel mechanism of autophagic cell death evoked by activation of the P2RX7 large pore specifically in dystrophic myoblasts [6]. http Stimulation of metabotropic P2Y receptors coupled with G q protein initiates a signalling pathway, involving an activation of phospholipase C and increase of IP3 concentration, which activates Ca 2+ release from the intracellular calcium stores in the sarcoplasmic reticulum.…”

Hsp90 regulates autophagy and plays a role in cancer therapy

“…Contrary to the abovementioned findings that GA treatment could reduce Beclin1 expression, dystrophic myoblasts showed a dose-dependent increase of Beclin1 in response to GA treatment [24]. As reported, the failure of proteasome and/ or chaperone machineries could cause aggresome formation [25].…”

“…As reported, the failure of proteasome and/ or chaperone machineries could cause aggresome formation [25]. An aggresome assay in dystrophic myoblasts shows that inhibition of Hsp90 by GA results in targeting of Beclin1 to aggresomes [24]. These indicate that in dystrophic myoblasts, Hsp90 inhibition could cause accumulation of nonfunctional Beclin1 in aggresomes.…”

Anti-inflammatory drugs for Duchenne muscular dystrophy: focus on skeletal muscle-releasing factors

“…P2X7 was substantially upregulated in skeletal muscle from mdx mice and in myoblasts isolated from DMD patients 81–85. Additionally, exposure of mdx myoblasts to extracellular ATP induced a significant increase in P2X7/pannexin1 channel-dependent Ca 2+ influx and release of interleukin (IL)-1β, suggesting that nucleotides released from dystrofic muscle can trigger inflammatory response in DMD through purinergic signaling 86.…”