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METTL14 Promotes Lipopolysaccharide-Induced Myocardial Damage via m6A-Dependent Stabilization of TRPM7 mRNA
Xinzhu Wu1, Pan Huang1, Yanmin Xiao1
1Department of Pediatrics, Puren Hospital, Wuhan University of Science and Technology.
Abstract:
Sepsis-induced myocardial injury (SIMI) is a vital pathological component of severe sepsis and septic shock. As a prevalent internal mRNA modification in eukaryotic cells, N6-methyladenosine (m6A) modification is implicated in sepsis and immune disorders. Methyltransferase-like 14 (METTL14), a core subunit of the methyltransferase complex that catalyzes messenger RNA m6A modification, is involved in the regulation of human cardiomyocyte cell line (AC16) injury. This study aimed to explore the role and mechanism of METTL14 in lipopolysaccharide (LPS) -induced myocardial injury.Cell viability and apoptosis were analyzed via 3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT), flow cytometry, and TdT-mediated dUTP nick-end labeling (TUNEL) assay. The Tumor necrosis factor alpha (TNF-α) and Interleukin-1beta (IL-1β) levels were analyzed via Enzyme linked immunosorbent assay (ELISA). Caspase-3 activity, reactive oxygen species activity, malondialdehyde level, and glutathione level were assessed using special assay kits. The levels of transient receptor potential melastatin 7 (TRPM7) and METTL14 mRNA were determined via Real-time quantitative polymerase chain reaction (RT-qPCR). Meanwhile, the protein levels of TRPM7, METTL14, phospho-p65 (p-p65), total p65 (p65), p-IκBα, and total IκBα (IκBα) were examined via western blot assay.LPS treatment repressed AC16 cell viability and induced cell apoptosis, inflammatory response, oxidative stress, and ferroptosis in vitro. METTL14 and TRPM7 were upregulated in LPS-treated AC16 cells. At the molecular level, METTL14 could increase the stability of TRPM7 mRNA via m6A methylation. Moreover, METTL14 deficiency could abolish LPS-triggered AC16 cell injury and ferroptosis via TRPM7 regulation.METTL14 knockdown reversed LPS-caused myocardial cell damage mainly by regulating the stability of TRPM7 mRNA, providing a novel therapeutic target for septic cardiomyopathy treatment.
Insights
Methyltransferase-like 14 (METTL14) plays a key role in sepsis-induced myocardial injury by stabilizing TRPM7 mRNA. METTL14 deficiency protects against lipopolysaccharide-induced heart cell damage and ferroptosis, offering a potential therapeutic target.
Area of Science:
- Cardiovascular Biology
- Molecular Biology
- Cellular Pathology
Background:
- Sepsis-induced myocardial injury (SIMI) is a critical complication of severe sepsis and septic shock.
- N6-methyladenosine (m6A) modification, regulated by METTL14, is involved in sepsis and immune responses.
- METTL14's role in lipopolysaccharide (LPS)-induced human cardiomyocyte (AC16) injury requires elucidation.
Purpose of the Study:
- To investigate the role and underlying mechanism of METTL14 in LPS-induced myocardial injury.
- To explore METTL14's regulation of transient receptor potential melastatin 7 (TRPM7) in cardiomyocytes.
- To assess METTL14's potential as a therapeutic target for septic cardiomyopathy.
Main Methods:
- Cell viability (MTT), apoptosis (flow cytometry, TUNEL), and inflammatory markers (ELISA) were assessed.
- Oxidative stress (ROS, MDA, GSH) and caspase-3 activity were measured.
- mRNA and protein levels of METTL14 and TRPM7 were analyzed using RT-qPCR and Western blot, respectively.
Main Results:
- LPS treatment induced AC16 cell injury, apoptosis, inflammation, oxidative stress, and ferroptosis.
- METTL14 and TRPM7 expression were upregulated in LPS-treated AC16 cells.
- METTL14 stabilized TRPM7 mRNA via m6A methylation, and METTL14 deficiency protected against LPS-induced injury and ferroptosis by regulating TRPM7.
Conclusions:
- METTL14 exacerbates LPS-induced myocardial injury and ferroptosis by enhancing TRPM7 mRNA stability.
- METTL14 knockdown mitigates cardiac cell damage and ferroptosis, suggesting METTL14 as a therapeutic target.
- Targeting METTL14 offers a novel strategy for treating septic cardiomyopathy.
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