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Autophagy-Related LC3 Accumulation Interacted Directly With LIR Containing RIPK1 and RIPK3, Stimulating Necroptosis in Hypoxic Cardiomyocytes

The exact relationships and detailed mechanisms between autophagy and necroptosis remain obscure. Here, we demonstrated the link between accumulated autophagosome and necroptosis by intervening with autophagic flux. We first confirmed that the LC3 interacting region (LIR) domain is present in the pr...

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Autores principales: Huang, Yao, Feng, Yanhai, Cui, Lin, Yang, Lei, Zhang, Qiong, Zhang, Junhui, Jiang, Xupin, Zhang, Xingyue, Lv, Yanling, Jia, Jie-Zhi, Zhang, Dong-Xia, Huang, Yue-Sheng
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8344065/
https://www.ncbi.nlm.nih.gov/pubmed/34368130
http://dx.doi.org/10.3389/fcell.2021.679637
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author Huang, Yao
Feng, Yanhai
Cui, Lin
Yang, Lei
Zhang, Qiong
Zhang, Junhui
Jiang, Xupin
Zhang, Xingyue
Lv, Yanling
Jia, Jie-Zhi
Zhang, Dong-Xia
Huang, Yue-Sheng
author_facet Huang, Yao
Feng, Yanhai
Cui, Lin
Yang, Lei
Zhang, Qiong
Zhang, Junhui
Jiang, Xupin
Zhang, Xingyue
Lv, Yanling
Jia, Jie-Zhi
Zhang, Dong-Xia
Huang, Yue-Sheng
author_sort Huang, Yao
collection PubMed
description The exact relationships and detailed mechanisms between autophagy and necroptosis remain obscure. Here, we demonstrated the link between accumulated autophagosome and necroptosis by intervening with autophagic flux. We first confirmed that the LC3 interacting region (LIR) domain is present in the protein sequences of RIPK1 and RIPK3. Mutual effects among LC3, RIPK1, and RIPK3 have been identified in myocardium and cardiomyocytes. Direct LC3-RIPK1 and LC3-RIPK3 interactions were confirmed by pull-down assays, and their interactions were deleted after LIR domain mutation. Moreover, after disrupting autophagic flux under normoxia with bafilomycin A1 treatment, or with LC3 or ATG5 overexpression adenovirus, RIPK1, RIPK3, p-RIPK3, and p-MLKL levels increased, suggesting necroptosis activation. Severe disruptions in autophagic flux were observed under hypoxia and bafilomycin A1 co-treated cardiomyocytes and myocardium and led to more significant activation of necroptosis. Conversely, after alleviating hypoxia-induced autophagic flux impairment with LC3 or ATG5 knockdown adenovirus, the effects of hypoxia on RIPK1 and RIPK3 levels were reduced, which resulted in decreased p-RIPK3 and p-MLKL. Furthermore, necroptosis was inhibited by siRNAs against RIPK1 and RIPK3 under hypoxia or normoxia. Based on our results, LIR domain mediated LC3-RIPK1 and LC3-RIPK3 interaction. Besides, autophagosome accumulation under hypoxia lead to necrosome formation and, in turn, necroptosis, while when autophagic flux was uninterrupted, RIPK1 and RIPK3 were cleared through an autophagy-related pathway which inhibited necroptosis. These findings provide novel insights for the role of LC3 in regulating cardiomyocyte necroptosis, indicating its therapeutic potential in the prevention and treatment of hypoxic myocardial injury and other hypoxia-related diseases.
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spelling pubmed-83440652021-08-07 Autophagy-Related LC3 Accumulation Interacted Directly With LIR Containing RIPK1 and RIPK3, Stimulating Necroptosis in Hypoxic Cardiomyocytes Huang, Yao Feng, Yanhai Cui, Lin Yang, Lei Zhang, Qiong Zhang, Junhui Jiang, Xupin Zhang, Xingyue Lv, Yanling Jia, Jie-Zhi Zhang, Dong-Xia Huang, Yue-Sheng Front Cell Dev Biol Cell and Developmental Biology The exact relationships and detailed mechanisms between autophagy and necroptosis remain obscure. Here, we demonstrated the link between accumulated autophagosome and necroptosis by intervening with autophagic flux. We first confirmed that the LC3 interacting region (LIR) domain is present in the protein sequences of RIPK1 and RIPK3. Mutual effects among LC3, RIPK1, and RIPK3 have been identified in myocardium and cardiomyocytes. Direct LC3-RIPK1 and LC3-RIPK3 interactions were confirmed by pull-down assays, and their interactions were deleted after LIR domain mutation. Moreover, after disrupting autophagic flux under normoxia with bafilomycin A1 treatment, or with LC3 or ATG5 overexpression adenovirus, RIPK1, RIPK3, p-RIPK3, and p-MLKL levels increased, suggesting necroptosis activation. Severe disruptions in autophagic flux were observed under hypoxia and bafilomycin A1 co-treated cardiomyocytes and myocardium and led to more significant activation of necroptosis. Conversely, after alleviating hypoxia-induced autophagic flux impairment with LC3 or ATG5 knockdown adenovirus, the effects of hypoxia on RIPK1 and RIPK3 levels were reduced, which resulted in decreased p-RIPK3 and p-MLKL. Furthermore, necroptosis was inhibited by siRNAs against RIPK1 and RIPK3 under hypoxia or normoxia. Based on our results, LIR domain mediated LC3-RIPK1 and LC3-RIPK3 interaction. Besides, autophagosome accumulation under hypoxia lead to necrosome formation and, in turn, necroptosis, while when autophagic flux was uninterrupted, RIPK1 and RIPK3 were cleared through an autophagy-related pathway which inhibited necroptosis. These findings provide novel insights for the role of LC3 in regulating cardiomyocyte necroptosis, indicating its therapeutic potential in the prevention and treatment of hypoxic myocardial injury and other hypoxia-related diseases. Frontiers Media S.A. 2021-07-23 /pmc/articles/PMC8344065/ /pubmed/34368130 http://dx.doi.org/10.3389/fcell.2021.679637 Text en Copyright © 2021 Huang, Feng, Cui, Yang, Zhang, Zhang, Jiang, Zhang, Lv, Jia, Zhang and Huang. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Cell and Developmental Biology
Huang, Yao
Feng, Yanhai
Cui, Lin
Yang, Lei
Zhang, Qiong
Zhang, Junhui
Jiang, Xupin
Zhang, Xingyue
Lv, Yanling
Jia, Jie-Zhi
Zhang, Dong-Xia
Huang, Yue-Sheng
Autophagy-Related LC3 Accumulation Interacted Directly With LIR Containing RIPK1 and RIPK3, Stimulating Necroptosis in Hypoxic Cardiomyocytes
title Autophagy-Related LC3 Accumulation Interacted Directly With LIR Containing RIPK1 and RIPK3, Stimulating Necroptosis in Hypoxic Cardiomyocytes
title_full Autophagy-Related LC3 Accumulation Interacted Directly With LIR Containing RIPK1 and RIPK3, Stimulating Necroptosis in Hypoxic Cardiomyocytes
title_fullStr Autophagy-Related LC3 Accumulation Interacted Directly With LIR Containing RIPK1 and RIPK3, Stimulating Necroptosis in Hypoxic Cardiomyocytes
title_full_unstemmed Autophagy-Related LC3 Accumulation Interacted Directly With LIR Containing RIPK1 and RIPK3, Stimulating Necroptosis in Hypoxic Cardiomyocytes
title_short Autophagy-Related LC3 Accumulation Interacted Directly With LIR Containing RIPK1 and RIPK3, Stimulating Necroptosis in Hypoxic Cardiomyocytes
title_sort autophagy-related lc3 accumulation interacted directly with lir containing ripk1 and ripk3, stimulating necroptosis in hypoxic cardiomyocytes
topic Cell and Developmental Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8344065/
https://www.ncbi.nlm.nih.gov/pubmed/34368130
http://dx.doi.org/10.3389/fcell.2021.679637
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