Abstract 17073: Accumulation of P62 by Dysregulation of Autophagy Induces Rip1-dependent Necroptosis in Cardiomyocytes: a Novel Interplay Between Autophagy and Necroptosis
Background: Necroptosis, a recently recognized form of cell death, has been implicated in various pathological conditions including myocardial infarction. Activation of receptor-interacting protein kinase (RIP) 1, leading to RIP1-RIP3 complex formation, is a trigger event of necroptosis, but the mechanism for RIP1 activation has not been characterized fully.
Objective: We examined the relationship between RIP1-dependent necroptosis and autophagy in cardiomyocytes.
Methods: In H9c2 cardiomyoblasts, the necroptotic pathway was activated using TNF-α (TNF, 50 ng/ml) alone or co-incubation of TNF-α with 20 μM z-VAD-fmk, a pan-caspase inhibitor (TNF/zVAD). As an index of necrosis, the amount of LDH released into the culture medium 24 h after serum withdrawal was expressed as a percentage of total LDH. Necrostatin-1 (Nec-1, 20 μM) and cyclosporine A (CsA, 1 μM) were used for inhibiting RIP1-dependent necroptosis and mitochondrial permeability transition pore opening, respectively.
Results: Treatment with TNF induced cleavage of RIP1 and caspase-3 and increased necrosis from 26.6±4.3% in the vehicle-treated control to 39.1±4.8%. Addition of zVAD to TNF abolished the TNF-induced cleavage of RIP1 and caspase-3 but paradoxically enhanced necrosis to 70.6±2.7%. This augmentation of TNF-induced necrosis by zVAD was completely prevented by Nec-1 (39.4±4.0%) but not by CsA (66.3±1.3%), indicating the occurrence of RIP1-dependent necroptosis in cardiomyocytes. Immunoblot analysis showed that TNF/zVAD increased levels of both LC3-II and p62 by 57% and 53% compared to the levels with TNF alone, respectively, suggesting suppression of autophagic flux. Furthermore, p62 physically interacted with RIP1, and the interaction was enhanced by TNF/zVAD. Pretreatment with 10 nM of rapamycin, a promoter of autophagic flux, suppressed TNF/zVAD-induced necrosis (49.4±2.0%) and augmentation of p62-RIP1 interaction.
Conclusion: A RIP1-dependent necroptotic program operates in cardiomyocytes, and the program is enhanced by inhibition of apoptosis. Accumulation of p62 by suppression of autophagy plays a crucial role in RIP1-dependent necroptosis, indicating a direct mechanistic link between autophagy and necroptosis.
Author Disclosures: M. Ogasawara: None. T. Yano: None. T. Miki: None. M. Tanno: None. A. Kuno: None. H. Kouzu: None. H. Murase: None. T. Tobisawa: None. S. Muratsubaki: None. K. Nishizawa: None. M. Mizuno: None. S. Ishikawa: None. T. Miura: None.
- © 2014 by American Heart Association, Inc.