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Necroptosis is crucially involved in severe cardiac pathological conditions, whereas efficient autophagic flux is essential for myocardial survival. However, whether impaired autophagosome processing contributes to myocardial necroptosis after ischemia/reperfusion (MI/R) is unknown. Here we demonstrate that autophagosome accumulation enhances MI/R triggered myocardial necroptosis in aged hearts.
Young (3-4 mon) and aged (22-24 mon) mice were subjected to MI/R in vivo. The isolated hearts of each group were perfused and subjected to MI/R in vitro and treated with necroptosis inhibitor necrostain-1 to restrain necroptosis. Aged mice were injected with metformin to restore autophagy flux.
Autophagosome clearance was impaired in aged hearts and autophagosome abundance was further increased after MI/R. In young hearts, the autophagy substrate Sequestosome1 (p62/SQSTMl) was increased in ischemia and was degraded in reperfusion, while aged hearts exhibited increased p62 at basal and further accumulation during MI/R because of disabled degradation. Aged mice subjected to MI/R endured greater myocardial necroptosis, as evidenced by reductions in lactate dehydrogenase (LDH) release and Evans blue dye (EBD) penetration. We found that overload of p62 conjugated with the RIP1 and promoted massive RIP1-RIP3 complex (necrosome) during MI/R. Necrosome phosphorylated MLKL and promoted the translocation of MLKL from cytoplasm to membrane, which eventually wrecked the cell membrane. Treatment of necrostain-1 inhibited the RIP1-RIP3-MLKL signaling, which ameliorated MI/R injury compare with vehicle in aged hearts. Down-regulation of p62 significantly reduced p62-RIP1-RIP3-MLKL signaling cascade in the aged hearts. Meanwhile, treatment of metformin restored the autophagy flux in the senescent hearts and decreased p62 level, which protected aged hearts from MI/R-induced RIP3-dependent necroptosis.
Our findings show that accumulation of p62 is sufficient to promote RIP1-RIP3-MLKL cascade by triggering the formation of p62-RIP1, thereby enhancing MI/R induced necroptosis in aged hearts. These findings confirm the new mode of autophagic dysfunction promoted necroptosis and provide new views for aging-related myocardial vulnerability.