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Silencing m6A Reader YTHDC1 reduces inflammatory response in sepsis-induced cardiomyopathy by inhibiting SERPINA3N expression
Abstract
Sepsis-induced cardiomyopathy (SIC) is one of the most common complications of infection-induced sepsis. An imbalance in inflammatory mediators is the main factor leading to SIC. N6-methyladenosine (m6A) is closely related to the occurrence and development of sepsis. m6A reader YTH Domain Containing 1 (YTHDC1) is an m6A N6-methyladenosine recognition protein. However, the role of YTHDC1 in SIC remains unclear. Herein, we demonstrated that YTHDC1-shRNA inhibits inflammation, reduces inflammatory mediators, and improves cardiac function in a lipopolysaccharide (LPS)-induced SIC mouse model. Based on the Gene Expression Omnibus (GEO) database analysis, serine protease inhibitor A3N (SERPINA3N) is a differential gene of SIC. Further, RNA immunoprecipitation (RIP) indicated that SERPINA3N mRNA can bind to YTHDC1, which regulates the expression of SERPINA3N. SERPINA3N-siRNA reduced LPS-induced inflammation of cardiac myocytes. In conclusion, the m6A reader YTHDC1 regulates SERPINA3N mRNA expression to mediate the levels of inflammation in SIC. Such findings add to the relationship between m6A reader YTHDC1 and SIC, providing a new research avenue for the therapeutic mechanism of SIC.
... By influencing the nuclear localization, stability, decay and splicing of RNA, Ythdc1 regulates a range of RNA metabolic reactions, thereby influencing disease development. Previous studies have suggested potential beneficial effects of Ythdc1 in cardiomyopathies 62,63 . For instance, deletion of Ythdc1 in mice was found to induce dilated cardiomyopathy, characterized by significant left ventricular enlargement, severe systolic function, reduced contractility of myocardial cell contractility and disorganized sarcomere arrangement. ...
The therapeutic efficacy of bone marrow mesenchymal stem cells (BMSCs) in myocardial infarction (MI) is hindered by poor cell survival. This study explored the role of N6-methyladenosine (m6A) regulation, specifically YTHDC1, in improving BMSC transplantation for MI. By screening m6A-related regulators in hypoxia and serum deprivation (HSD)-induced BMSC apoptosis, YTHDC1 was found to be downregulated. Overexpression of Ythdc1 in BMSCs reduced apoptosis markers, reactive oxygen species (ROS) release, and improved cell survival under HSD conditions. Conversely, Ythdc1 knockdown enhanced apoptosis. In rat MI models, transplantation of Ythdc1-overexpressing BMSCs improved cardiac function and reduced myocardial fibrosis. Mechanistically, YTHDC1 interacts with nuclear factor kappa B (NF-κB) inhibitor-alpha mRNA, suggesting its involvement in BMSC survival pathways. This study identifies YTHDC1 as a potential target to enhance BMSC efficacy in MI therapy.
... Xie et al. used RNA m6A dot blot to detect elevated m6A levels in the mouse myocardial tissues of the LPS group compared to the control group and identified YTHDC1 as the primary upregulated m6A reader protein. [21] Through GEO database analysis, SERPINA3N was found to be significantly upregulated in septic mouse and rat myocardial tissues. Using knockout methods, it was confirmed that YTHDC1 regulates the inflammatory response of SIC through m6A modification in SERPINA3N transcripts. ...
Sepsis-induced cardiomyopathy (SIC) is a common and high-mortality complication among critically ill patients. Uncertainties persist regarding the pathogenesis, pathophysiology, and diagnosis of SIC, underscoring the necessity to investigate potential biological mechanisms. With the rise of omics technologies, leveraging their high throughput and big data advantages, a systems biology perspective is employed to study the biological processes of SIC. This approach aids in gaining a better understanding of the disease’s onset, progression, and outcomes, ultimately providing improved guidance for clinical practices. This review summarizes the currently applied omics technologies, omics studies related to SIC, and relevant omics databases.
... Of note, YTHDC1 is the main m(6)A nuclear reader that has been implicated in various biological processes [16], including inflammatory diseases. YTHDC1 downregulation suppresses inflammation in sepsis-induced cardiomyopathy [17]. Another study reveals that YTHDC1 is crucial to maintain islet β-cell function, deficiency of which results in diabetes [18]. ...
Background
YTHDC1, a key m(6)A nuclear reader, plays a crucial role in regulating mRNA splicing, export, and stability. However, the functional significance and regulatory mechanisms of YTHDC1 in inflammatory bowel disease (IBD) remain to be explored.
Methods
We established a dextran sulfate sodium (DSS)-induced murine colitis model in vivo and LPS/IFN-γ-stimulated macrophage inflammation in vitro. The expression of YTHDC1 was determined. Colocalization of YTHDC1 and macrophages was assayed by immunofluorescence staining. LV-YTHDC1 or shYTHDC1 lentiviruses were applied for YTHDC1 overexpression or inhibition. For NF-κB inhibition, JSH-23 was utilized. The interaction of YTHDC1 and Beclin1 mRNA was determined by RIP, and the m6A modification of Beclin1 was confirmed by MeRIP.
Results
In DSS-induced colitis and LPS/IFN-γ-treated RAW264.7 macrophages, we observed a significant downregulation of YTHDC1. Overexpression of YTHDC1 resulted in decreased levels of iNOS, CD86, and IL-6 mRNA, along with inhibited NF-κB activation in LPS/IFN-γ-treated RAW264.7 cells. Conversely, downregulation of YTHDC1 promoted iNOS expression and inhibited autophagy. Additionally, the effect of YTHDC1 knockdown on CD86 and IL-6 mRNA induced by LPS/IFN-γ was abolished by the NF-κB inhibitor JSH-23. Mechanistically, YTHDC1 interacted with Beclin1 mRNA, thereby stabilizing Beclin1 mRNA and enhancing Beclin1 expression and autophagy. These effects ultimately led to the inhibition of NF-κB signaling in LPS/IFN-γ-challenged macrophages.
Conclusions
YTHDC1 inhibited the macrophage-mediated inflammatory response by stabilizing Beclin1 mRNA, which may be a potential therapeutic target for the treatment of IBD.
... Emerging evidence has unravelled a close relationship between septic cardiomyopathy and m 6 A modification, which is relevant to sepsis-induced immune responses and subsequent cardiac injuries [14,21,22]. In this work, our findings reveal that the m 6 Septic cardiomyopathy is one subtype of severe sepsis with a series of pathophysiological changes such as inflammation activation, myocardial dysfunction, metabolic disturbance, microcirculatory disturbance, and so forth [1,23]. ...
Introduction
Septic cardiomyopathy is a sepsis-mediated cardiovascular complication with severe microcirculatory malperfusion. Emerging evidence has highlighted the protective effects of pulsatile flow in case of microcirculatory disturbance, yet the underlying mechanisms are still elusive. The objective of this study was to investigate the mechanisms of N6-methyladenosine (m ⁶ A) modification in the alleviation of septic cardiomyopathy associated with extracorporeal membrane oxygenation (ECMO)-generated pulsatile flow.
Methods
Rat model with septic cardiomyopathy was established and was supported under ECMO either with pulsatile or non-pulsatile flow. Peripheral perfusion index (PPI) and cardiac function parameters were measured using ultrasonography. Dot blot assay was applied to examine the m ⁶ A level, while qRT-PCR, Western blot, immunofluorescence, and immunohistochemistry were used to measure the expressions of related genes. RNA immunoprecipitation assay was performed to validate the interaction between molecules.
Results
The ECMO-generated pulsatile flow significantly elevates microcirculatory PPI, improves myocardial function, protects the endothelium, and prolongs survival in rat models with septic cardiomyopathy. The pulsatile flow mediates the METTL14-mediated m ⁶ A modification to zonula occludens- (ZO-) 1 mRNA which stabilizes the ZO-1 mRNA depending on the presence of YTHDF2. The pulsatile flow suppresses the PI3K-Akt signaling pathway, of which the downstream molecule Foxo1, a negative transcription factor of METTL14, binds to the METTL14 promoter and inhibits the METTL14-induced m ⁶ A modification.
Conclusion
The ECMO-generated pulsatile flow increases METTL14-induced m ⁶ A modification in ZO-1 and attenuates the progression of septic cardiomyopathy, suggesting that pulsatility might be a new therapeutic strategy in septic cardiomyopathy by alleviating microcirculatory disturbance.
Myasthenia gravis (MG), a rare autoimmune disorder, presents a complex pathogenesis involving various immune molecules. The modification of N6-methyladenosine (m6A) regulates diverse immune metabolic and immunopathological processes; however, its role in MG remains unclear. We downloaded dataset GSE85452 from the GEO database to identify differentially expressed genes regulated by m6A. The Random Forest (RF) method was utilized to identify pivotal regulatory genes associated with m6A modification. Subsequently, a prognostic model was crafted and confirmed using this gene set. Patients with MG were stratified according to the expression levels of these key regulatory genes. Additionally, MG-specific immune signatures were delineated by examining immune cell infiltration patterns and their correlations. Further functional annotation, protein-protein interaction mapping, and molecular docking analyses were performed on these immune biomarkers, leading to the discovery of three genes that exhibited significant differential expression within the dataset: RBM15, CBLL1, and YTHDF1.The random forest algorithm confirmed these as key regulatory genes of m6A in MG, validated by constructing a clinical prediction model. Based on key regulatory gene expression, we divided MG patients into two groups, revealing two distinct m6A modification patterns with varying immune cell abundances. We also discovered 61 genes associated with the m6A phenotype and conducted an in-depth exploration of their biological roles. RBM15, CBLL1, and YTHDF1 were found positively correlated with CD56dim natural killer cells, natural killer T cells, and type 1 helper T cells. These genes were stable diagnostic m6A-related markers in both discovery and validation cohorts. Our findings suggest RBM15, CBLL1, and YTHDF1 as immune markers for MG. Further analysis of these genes may elucidate their roles in the immune microenvironment of MG.
Neuroinflammation, blood–brain barrier (BBB) dysfunction, neuron and glia injury/death and myelin damage are common central nervous system (CNS) pathologies observed in various neurological diseases and injuries. Serine protease inhibitor (Serpin) clade A member 3n (Serpina3n), and its human orthologue SERPINA3, is an acute‐phase inflammatory glycoprotein secreted primarily by the liver into the bloodstream in response to systemic inflammation. Clinically, SERPINA3 is dysregulated in brain cells, cerebrospinal fluid and plasma in various neurological conditions. Although it has been widely accepted that Serpina3n/SERPINA3 is a reliable biomarker of reactive astrocytes in diseased CNS, recent data have challenged this well‐cited concept, suggesting instead that oligodendrocytes and neurons are the primary sources of Serpina3n/SERPINA3. The debate continues regarding whether Serpina3n/SERPINA3 induction represents a pathogenic or a protective mechanism. Here, we propose possible interpretations for previously controversial data and present perspectives regarding the potential role of Serpina3n/SERPINA3 in CNS pathologies, including demyelinating disorders where oligodendrocytes are the primary targets. We hypothesise that the ‘good’ or ‘bad’ aspects of Serpina3n/SERPINA3 depend on its cellular sources, its subcellular distribution (or mis‐localisation) and/or disease/injury types. Furthermore, circulating Serpina3n/SERPINA3 may cross the BBB to impact CNS pathologies. Cell‐specific genetic tools are critically important to tease out the potential roles of cell type‐dependent Serpina3n in CNS diseases/injuries.
This study aimed to investigate the role of the N6-methyladenosine (m ⁶ A) reader protein YTHDC1 in heart development and its potential molecular mechanisms. Animal experiments were conducted using cardiac-specific Ythdc1 knockout ( Ythdc1- CKO) mice, and human heart samples were collected from aborted fetuses. Echocardiography, immunoblotting, RNA-Seq, and ATAC-Seq were performed to assess cardiac function, gene expression, and chromatin accessibility. The results revealed that YTHDC1 expression was highest during embryonic and early postnatal stages and gradually decreased with age. Cardiac-specific deletion of Ythdc1 resulted in abnormal heart development, early dilated cardiomyopathy, and severe heart failure. RNA-Seq analysis revealed significant changes in gene expression profiles, particularly genes related to cardiac contraction and transmembrane transport. ATAC-Seq analysis demonstrated significant changes in chromatin accessibility, and the binding motifs of the transcription factors Mef2a, Mef2b, Mef2c, and Mef2d, which are essential for cardiac development, were switched off in Ythdc1- CKO mouse hearts. In conclusion, this study demonstrates that YTHDC1 plays a critical role in heart development and its deficiency leads to abnormal cardiac development and function. The findings provide insights into the molecular mechanisms underlying heart development and suggest potential therapeutic targets for heart diseases.
N6-methyladenosine (m6A) modification is a class of epitope modifications that has received significant attention in recent years, particularly in relation to its role in various diseases, including sepsis. Epigenetic research has increasingly focused on m6A modifications, which is influenced by the dynamic regulation of three protein types: ‟Writers” (such as METTL3/METTL14/WTAP)—responsible for m6A modification; ‟Erasers” (FTO and ALKBH5)—involved in m6A de-modification; and ‟Readers” (YTHDC1/2, YTHDF1/2/3)—responsible for m6A recognition. Sepsis, a severe and fatal infectious disease, has garnered attention regarding the crucial effect of m6A modifications on its development. In this review, we attempted to summarize the recent studies on the involvement of m6A and its regulators in sepsis, as well as the significance of m6A modifications and their regulators in the development of novel drugs and clinical treatment. The potential value of m6A modifications and modulators in the diagnosis, treatment, and prognosis of sepsis has also been discussed.
Graphical abstract
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