Suppression of autophagic flux contributes to cardiomyocyte death by activation of necroptotic pathways

Abstract Background The role of necroptosisin myocardial injury has not been fully characterized. Here we examined roles of mitochondrial permeability transition pore( mPTP) and autophagy in necroptosisof cardiomyocytes. Methods and results In H9c2 cells, necroptosiswas induced by treatment with TNF-α (TNF) and z-VAD-fmk (zVAD) for 24 h, and necroptotic death was determined by LDH release (as % of total). TNF/zVAD increased LDH release from 16.6 ± 4.3% to 60.6 ± 2.7%, and the LDH release was suppressed by necrostatin-1 (29.4 ± 4.0%), a RIP1 inhibitor, and by siRNA-mediated knockdown of RIP3 (27.7 ± 2.0%), confirming RIP1-RIP3-dependent necroptosis. TNF/zVAD-induced necroptosiswas not attenuated by mPTPinhibitors or GSK-3β inhibitors. TNF/zVAD increased LC3-II level, but the change was not further enhanced by bafilomycinA1. The increase of LC3-II by TNF/zVAD was associated with suppression of both autophagic flux and LC3- LAMP1co-localization. TNF/zVAD did not modify phosphorylation of Akt, p70s6K, AMPK, ULK1or VASP but significantly increased RIP1-p62 binding and conversely reduced p62-LC3 binding. Rapamycin inhibited RIP1-p62 and RIP1-RIP3 interactions induced by TNF/zVAD and partly restored autophagic flux and suppressed LDH release in TNF/zVAD-treated cells. The effect of rapamycin on LDH release was reduced by knockdown of Atg5expression. Knockdown of p62 by siRNA augmented LDH release by TNF/zVAD. Conclusion Suppression of autophagic flux contributes to RIP1-RIP3 interaction and necroptosisof cardiomyocytes, and sequestration of p62 from its interaction with LC3-II by p62-RIP1 interaction possibly underlies the suppressed autophagy. The mPTPis unlikely to play a major role in execution of necroptosisin cardiomyocytes.