Abstract
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.
Highlights
Heart, an essential component of circulation system, plays an important role in maintaining life activity
ATG5-OE resulted in increased levels of p-RIPK3, mixed lineage kinase domain-like pseudokinase (MLKL) and phosphorylated MLKL (p-MLKL), and enhanced cytotoxicity in cardiomyocytes, while ATG5-KD lowered levels of p-RIPK3, MLKL and p-MLKL and lower cytotoxicity in cardiomyocytes under hypoxia (Figures 6A–C,E–G, p < 0.05). These results further suggested that autophagosome accumulations induced by ATG5 overexpression or hypoxia were able to stimulate necroptosis, and decreased autophagosome mediated by ATG5 knockdown inhibited necroptosis, which was reflected in the microtubule-associated protein 1A/1Blight chain 3 (LC3)-associated decrease in RIPK1 and RIPK3 levels
The salient findings presented here revealed that direct interactions existed between LC3-RIPK1 and LC3-RIPK3 through LC3 interacting region (LIR) domain
Summary
An essential component of circulation system, plays an important role in maintaining life activity. The pigment epitheliumderived factor (PEDF) is shown to activate necroptosis by regulating RIPK3 in hypoxic cardiomyocytes; the detailed mechanisms of hypoxia-induced necrosome formation are still poorly understood (Gao et al, 2015). Receptor-interacting protein kinase (RIPK) 1 (RIPK1, known as RIP1) was the first discovered molecule in the necroptosis pathway and has been identified as a component of the death complex (i.e., necrosome) (Lee et al, 2004; Mompeán et al, 2018). Upon inhibition of the Fasassociated death domain (FADD) or caspase activity by genetic or chemical methods, RIPK1 and RIPK3 ( known as RIP3) form the necrosome through the homotypic interaction motif (RHIM) domains. Determining the mechanisms underlying necrosome formation in hypoxic heart is important in preventing myocardial injury caused by hypoxia
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