Abstract
Previous studies have demonstrated that cardiomyocyte apoptosis, ferroptosis, and inflammation participate in the progress of sepsis-induced cardiomyopathy (SIC). Although Islet cell autoantigen 69 (ICA69) is an imperative molecule that could regulate inflammation and immune response in numerous illnesses, its function in cardiovascular disease, particularly in SIC, is still elusive. We confirmed that LPS significantly enhanced the expression of ICA69 in wild-type (WT) mice, macrophages, and cardiomyocytes. The knockout of ICA69 in lipopolysaccharide(LPS)-induced mice markedly elevated survival ratio and heart function, while inhibiting cardiac muscle and serum inflammatory cytokines, reactive oxygen (ROS), and ferroptosis biomarkers. Mechanistically, increased expression of ICA69 triggered the production of STING, which further resulted in the production of intracellular lipid peroxidation, eventually triggering ferroptosis and heart injury. Intriguingly, ICA69 deficiency only reversed the ferroptotic marker levels, such as prostaglandin endoperoxide synthase 2 (PTGS2), malonaldehyde (MDA), 4-hydroxynonenal (4HNE), glutathione peroxidase 4 (GPX4), superoxide dismutase (SOD), iron and lipid ROS, but had no effects on the xCT-dependent manner. Additionally, greater ICA69 level was identified in septic patients peripheralblood mononuclear cells (PBMCs) than in normal control groups. Generally, we unveil that ICA69 deficiency can relieve inflammation and ferroptosis in LPS-induced murine hearts and macrophages, making targeting ICA69 in heart a potentially promising treatment method for SIC.
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Introduction
Sepsis is a systemic inflammatory response that is accompanied by multiple organ dysfunction, oxidative stress, and overmuch inflammatory cytokines [1] sepsis-induced cardiomyopathy (SIC) is one of the common and well-elucidated complications in sepsis and sepsis-induced shock, while Gram-negative bacterial endotoxin (Lipopolysaccharide, LPS) serves as a key sepsis mediator for septicemia-associated multiple organ dysfunction or mortality [2].
Encoded by the ICA1 gene, ICA69 has a limited cellular distribution and tissue distribution. Past study primarily highlighted the physiopathological function of ICA69 in organ-specific autoimmune illnesses including Type 1 diabetes (T1D) [3]. Thymic deletion of ICA69 expression, for instance, is adequate to give rise to inflammatory events in various organs [4]. Nevertheless, our research revealed that ICA69 was notably regulated upward in lipopolysaccharide-induced cardiac tissue. According to previous research findings, ICA69 enrichment occurs in the proximity of the Golgi complex and its N-terminal half involves a BAR domain, a component that could bend or bind membranes and has mutual effect with phosphatide [5]. And the BAR-domain family encompasses numerous constituents, the majority of which function in transport and endocytosis [6].
STING, composed of multiple assumed transmembrane regions, is primarily anchored as a homodimer in the ER membrane in resting conditions [7]. Recent work suggests that, after STING binds cGAMP, it transfers to the ER-Golgi intermedium compartment (ERGIC) and the Golgi via the process depending on the COPI (coat protein complex I), COPII complex and ARF GTPases [8, 9], which is essential for the phosphorylation of STING and subsequent IRF3 stimulation [10, 11]. As STING marks an imperative molecule that can modulate inflammation and defense response in SIC, and participates in septic heart damage via inducing cardiac muscle cell pyroptosis [12], these results hint that ICA69 may participate in STING-dependent innate immune response.
Ferroptosis is a ROS-related and iron-related cell death, which is crucial for organ damage and target treatment of tumors [13, 14]. Recent studies show that the ferroptosis induction via high-iron diet or Gpx4 consumption stimulates the STING-related DNA sensor pathway, which finally causes the infiltration of macrophagus and pancreatic tumorigenesis [Assessment of oxidative stress our team carried out the specimen preparation as per the assay kit specification. The levels of malondialdehyde (MDA) [53], superoxide dismutase (SOD) [54], GSH/GSSG [55], total antioxidant capacity (T-AOC) [56] in serum and heart samples were measured via colorimetric determination by assay kits according to previous studies mentioned above. The results of MDA, SOD were expressed as a unit per mg protein (U/mg prot). The tissue weight was accurately weighed, while the homogenate was mechanically prepared in an ice bath at 2500 rpm for 10 min to produce a 10% supernatant. After the sample is prepared, the protein level can be detected by the BCA Protein Concentration Assay Kit to facilitate the subsequent calculation of Fe content in tissues or cells per unit protein weight. Afterwards, iron standard test sample and iron test base liquid were added in sequence. Eventually, we mix the well, while detecting the standard well at 562 nm with the enzyme plate analyzer, measuring the well absorbance, and finish the colorimetry within 1 h. The quantity of cardiac tissue creatine kinase isoenzyme (CK-MB) was determined by a biochemical analyzing machine automatically (ADVIA® 2400, Siemens Ltd., China). The enzyme activities of lactate dehydrogenase (LDH) in serum were identified by rapid and sensitive assay kits according to the instruction. Briefly, we produced the specimens for the standard curve by nicotinamide adenine dinucleotide mother liquor and LDH buffer. A 50 μL Reaction Mix with 48 μL LDH Assay Buffer in it and 2 μL LDH Substrate Mix was supplemented into the specimens or standard specimens for a whole hour at 37 °C free of light, producing a 450 nm absorbance. The extra mice in all groups (n = 10) were raised to study the survival status. The mortality was daily documented at the identical time node, while the survival rate was computed within seven days posterior to LPS injection at 10 mg/kg or PBS. Echocardiography was implemented by a Vevo 3100 ultrasonic equipment with a 10-MHz linear array ultrasound transducer (Fujifilm, VisualSonics, USA) after mice were anesthetized by 1.5% isoflurane. As the medial echocardiographic readings were collected from 3–5 heart cycles, the heart function indexes, such as fractional shortening (FS), heart rate (bpm), ejection fraction (EF), etc., were documented. The entire measured data here were depicted by average ± SEM or characteristic images of 1 representative from 3 separate assays. As GraphPad Prism 8.0.2 software for Windows was adopted for statistic observation, the comparison between the two groups was performed by Student’s t-test, and the diversities between the groups were compared by two-way ANOVA and corrected by Bonferroni, with survival condition assessed by Kaplan–Meier analysis. Human data were studied by Wilcoxon (Exact) rank-sum test, while association among the expression of ICA1 in PBMCs and Acute Physiology and Chronic Health Evaluation II value of septic cases was evaluated by the Pearson correlation analysis. A P < 0.05 was deemed as significant on statistics.Iron in serum and cardiac tissue
LDH and CK MB in cardiac tissue
Survival condition
Echocardiography
Statistical analysis
Data availability
The datasets adopted in our research are accessible from the relevant author on reasonable request.
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Acknowledgements
This research was supported by the Natural Science Foundation of China (Grant No. 81704180 and 81774109). This work was also supported in part by the Zhejiang Provincial Natural Science Foundation (Grant No. LY19H290008) and Wenzhou Science & Technology Bureau Foundation (No. 2018ZY003, Y20190108, and No. Y20190202). The authors also thank the Laboratory Animal Research Center and the Institute of Translational Medicine in Wenzhou Medical University for technical assistance. Our research was backed by the National Natural Science Foundation of China (Grants 81704180 and 81774109); the Zhejiang Provincial Natural Science Foundation (Grant No. LY19H290008) and Wenzhou Municipal Science and Technology Bureau (No. 2018ZY003, Y20190108, and No. Y20190202).
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J.W., C.K., and X.N. designed the whole research, analyzed all results, and wrote the manuscript. J.W. provided technical and material support. A.Z. and Y.Z. participated in most of the experiments. Q.D., Y.M., and S.L. performed the development of methodology and writing, review, and revision of the paper. F.L., Y.L., J.Z., and X.Y. helped in performing experiments. All authors read and approved the final paper.
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On the ethics review, human study (ChiCTR-INR-2100051426) was accepted by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University and was conformed to the protocols in the Declaration of Helsinki and in the Federal Policy for the Protection of Human Subjects, with informed consent provided by each participated patient. Each animal assay, aiming to bring the mouse killing to the minimum, was accepted by the Animal Experimentation Ethics Committee (Approval Ethical Inspection ID: wydw2019-0559).
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Kong, C., Ni, X., Wang, Y. et al. ICA69 aggravates ferroptosis causing septic cardiac dysfunction via STING trafficking. Cell Death Discov. 8, 187 (2022). https://doi.org/10.1038/s41420-022-00957-y
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DOI: https://doi.org/10.1038/s41420-022-00957-y
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