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Melatonin Alleviates LPS-Induced Pyroptotic Cell Death in Human Stem Cell-Derived Cardiomyocytes by Activating Autophagy.

ABSTRACT: Endotoxemia in sepsis remains a problem due to a lack of effective strategies. Our previous studies have demonstrated that melatonin (Mel) protects against ischemic heart injury and arteriosclerosis. However, its role in endotoxemia-exposed cardiomyocytes remains poorly understood. This study explored, for the first time, the protective effect of Mel on the pyroptosis of human stem cell-derived cardiomyocytes (hiPSC-CMs) exposed to lipopolysaccharide (LPS). Our results showed that treatment with 1 μM or 10 μM Mel for 12 h significantly improved 1 μg/ml LPS-induced hiPSC-CM injuries, as reflected by drastically decreased LDH release and increased cell viability, which was accompanied by the overt induction of autophagy. Specifically, Mel profoundly alleviated LPS-induced cell pyroptosis, as evidenced by decreased propidium iodide (PI) and active caspase-1 double-positive cell rates; suppressed the expression of NLRP3, cleaved caspase-1 (activated form of caspase-1), and GSDMD-NT (functional N-terminal fragment of GSDMD) expression; and inhibited the production of the cleaved IL-1β and cleaved IL-18 cytokines. Additionally, double-membrane autophagosomes were observed in LPS-injured hiPSC-CMs treated with 1 μM or 10 μM Mel. The hiPSC-CMs treated with LPS exhibited considerably fewer acidic vesicles (as revealed by LAMP1 staining) and autophagosomes (as revealed by LC3-II staining); however, Mel reversed this outcome in a dose-dependent manner. Furthermore, coincubation with rapamycin (an autophagy activator) or 3-MA (an autophagy inhibitor) accentuated and attenuated the antipyroptotic actions of Mel, respectively. Collectively, our findings demonstrate that Mel shields hiPSC-CMs against pyroptosis during endotoxemia by activating autophagy.

SUBMITTER: Qiu Y

PROVIDER: S-EPMC8643260 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:DNA damage regulated autophagy modulator 1 (DRAM1) is a stress-inducible regulator of autophagy and cell death. DRAM1 has been implicated in cancer, myocardial infarction, and infectious diseases, but the molecular and cellular functions of this transmembrane protein remain poorly understood. Previously, we have proposed DRAM1 as a host resistance factor for tuberculosis (TB) and a potential target for host-directed anti-infective therapies. In this study, we generated a zebrafish dram1 mutant and investigated its loss-of-function effects during Mycobacterium marinum (Mm) infection, a widely used model in TB research. In agreement with previous knockdown analysis, dram1 mutation increased the susceptibility of zebrafish larvae to Mm infection. RNA sequencing revealed major effects of Dram1 deficiency on metabolic, immune response, and cell death pathways during Mm infection, and only minor effects on proteinase and metabolic pathways were found under uninfected conditions. Furthermore, unchallenged dram1 mutants did not display overt autophagic defects, but autophagic targeting of Mm was reduced in the absence of Dram1. The phagocytic ability of macrophages in dram1 mutants was unaffected, but acidification of Mm-containing vesicles was strongly reduced, indicating that Dram1 is required for phagosome maturation. By in vivo imaging, we observed that Dram1-deficient macrophages fail to restrict Mm during early stages of infection. The resulting increase in bacterial burden could be reverted by knockdown of inflammatory caspase a (caspa) and gasdermin Eb (gsdmeb), demonstrating pyroptosis as the mechanism underlying premature cell death of Mm-infected macrophages in dram1 mutants. Collectively, these data demonstrate that dissemination of mycobacterial infection in zebrafish larvae is promoted in the absence of Dram1 due to reduced maturation of mycobacteria-containing vesicles, failed intracellular containment, and consequent pyroptotic death of infected macrophages. These results provide new evidence that Dram1 plays a central role in host resistance to intracellular infection, acting at the crossroad of autophagy and cell death.

Project description:Autophagy is a highly conserved intracellular process that preserves cellular homeostasis by mediating the lysosomal degradation of virtually any component of the cytoplasm. Autophagy is a key instrument of cellular response to several stresses, including endoplasmic reticulum (ER) stress. Cancer cells have developed high dependency on autophagy to overcome the hostile tumor microenvironment. Thus, pharmacological activation or inhibition of autophagy is emerging as a novel antitumor strategy. ERK5 is a novel member of the MAP kinase family that is activated in response to growth factors and different forms of stress. Recent work has pointed ERK5 as a major player controlling cancer cell proliferation and survival. Therefore small-molecule inhibitors of ERK5 have shown promising therapeutic potential in different cancer models. Here, we report for the first time ERK5 as a negative regulator of autophagy. Thus, ERK5 inhibition or silencing induced autophagy in a panel of human cancer cell lines with different mutation patterns. As reported previously, ERK5 inhibitors (ERK5i) induced apoptotic cancer cell death. Importantly, we found that autophagy mediates the cytotoxic effect of ERK5i, since ATG5-/- autophagy-deficient cells viability was not affected by these compounds. Mechanistically, ERK5i stimulated autophagic flux independently of the canonical regulators AMPK or mTORC1. Moreover, ERK5 inhibition resulted in ER stress and activation of the Unfolded Protein Response (UPR) pathways. Specifically, ERK5i induced expression of the ER luminal chaperone BiP (a hallmark of ER stress), the UPR markers CHOP and ATF4, and the spliced form of XBP1. Pharmacological inhibition of UPR with chemical chaperone TUDC, or ATF4 silencing, resulted in impaired ERK5i-mediated UPR, autophagy and cytotoxicity. Overall, our results suggest that ERK5 inhibition induces autophagy-mediated cancer cell death by activating ER stress. Since ERK5 inhibition sensitizes cancer cells and tumors to chemotherapy, future work will determine the relevance of UPR and autophagy in the combined use of chemotherapy and ERK5i to tackle Cancer.

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Melatonin Alleviates LPS-Induced Pyroptotic Cell Death in Human Stem Cell-Derived Cardiomyocytes by Activating Autophagy.

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