Purpose: Necroptosis, a programmed cell death, has been implicated in various pathological conditions including heart failure. Here we examined whether and how receptor-interacting protein kinase (RIP) 1-dependent necroptotic signal modulates autophagy in cardiomyocytes, focusing on two modulators of autophagy, p62 and p53.
Methods and Results: In H9c2 cells, the necroptotic pathway was activated by co-incubation with TNF (50 ng/ml) and z-VAD-fmk (zVAD, 20 μM), a pan-caspase inhibitor. TNF/zVAD increased LDH in the culture medium, which was prevented by necrostatin-1, a RIP1 inhibitor, and attenuated by rapamycin, a promoter of autophagy. The protective effect of rapamycin was blocked by Atg5 knockdown, confirming role of autophagy in the protection. TNF/zVAD did not affect phosphorylation of protein kinases that inhibit autophagy (Akt and p70S6K, a kinase downstream of mTORC1) and/or activate autophagy (AMPK, ULK1 and VASP, a target downstream of PKA). However, TNF/zVAD suppressed autophagic flux that was assessed by monitoring autophagosomes and autophysosomes using transfection of RFP-GFP-LC3. TNF/zVAD increased protein level of p53 by 2.6 fold, and immunofluorescence experiments revealed that upregulated p53 was localized in the nucleus. However, extent of TNF/zVAD-induced cell death was not changed by siRNA-mediated knockdown of p53 or up-regulation of p53 by Nutlin-3 (10 μM), a MDM2 inhibitor. Immunoprecipitation experiments revealed that TNF/zVAD reduced interaction between LC3-II and p62, a critical event in autophagosome formation and conversely increased interaction between RIP1 and p62, suggesting that p62 was sequestered by RIP1 from LC3-II-p62 complex formation. siRNA-mediated knockdown of p62 exaggerated TNF/zVAD-induced cell death. The changes in p62 interactosomes by TNF/zVAD were reversed by pretreatment with rapamycin.
Conclusion: Cardiomyocyte necroptosis is associated with suppression of autophagy in which RIP1-mediated sequestration of p62 from LC3-II-p62 complexes formation is involved. Up-regulation of p53 by activation of the necroptotic pathway does not directly contribute to cell death, though it is possibly involved in modulation of metabolism.