METTL3 boosts mitochondrial fission and induces cardiac fibrosis by enhancing LncRNA GAS5 methylation.

Dysregulated mitochondrial metabolism occurs in several pathological processes characterized by cell proliferation and migration. Nonetheless, the role of mitochondrial fission is not well appreciated in cardiac fibrosis, which is accompanied by enhanced fibroblast proliferation and migration. We investigated the causes and consequences of mitochondrial fission in cardiac fibrosis using cultured cells, animal models, and clinical samples. Increased METTL3 expression caused excessive mitochondrial fission, resulting in the proliferation and migration of cardiac fibroblasts that lead to cardiac fibrosis. Knockdown of METTL3 suppressed mitochondrial fission, inhibiting fibroblast proliferation and migration for ameliorating cardiac fibrosis. Elevated METTL3 and N6-methyladenosine (m6A) levels were associated with low expression of long non-coding RNA GAS5. Mechanistically, METTL3-mediated m6A methylation of GAS5 induced its degradation, dependent of YTHDF2. GAS5 could interact with mitochondrial fission marker Drp1 directly; overexpression of GAS5 suppressed Drp1-mediated mitochondrial fission, inhibiting cardiac fibroblast proliferation and migration. Knockdown of GAS5 produced the opposite effect. Clinically, increased METTL3 and YTHDF2 levels corresponded with decreased GAS5 expression, increased m6A mRNA content and mitochondrial fission, and increased cardiac fibrosis in human heart tissue with atrial fibrillation. We describe a novel mechanism wherein METTL3 boosts mitochondrial fission, cardiac fibroblast proliferation, and fibroblast migration: METTL3 catalyzes m6A methylation of GAS5 methylation in a YTHDF2-dependent manner. Our findings provide insight into the development of preventative measures for cardiac fibrosis.

IGFBP3 epigenetic promotion induced by METTL3 boosts cardiac fibroblast activation and fibrosis.

Cardiac fibrosis remains an unresolved problem in heart disease. Its etiology is directly caused by the activation and proliferation of cardiac fibroblasts (CFs). However, there is limited information regarding the biological role of cardiac fibroblasts in cardiac fibrosis. Herein, we screened out a gene, IGFBP3, whose expression significantly increased in TGF-ß1-stimulated human primary CFs by mining RNA-Seq data for differential and WGCNA. We verified the IGFBP3's expression in transverse aortic constriction (TAC) surgery, isoproterenol (ISO)-induced cardiac fibrosis models, and TGFß1-stimulated mouse primary CFs. We also found that the knockdown of IGFBP3 could inhibit the migration and proliferation ability of CFs. Furthermore, we found that aberrant N6-methyladenosine(m6A) mRNA modifications in the animal model and activated CFs may regulate the expression of IGFBP3 in developing cardiac fibrosis. Silencing METTL3 could downregulate the expression of IGFBP3 and inhibit the activation of CFs and the degree of cardiac fibrosis both in vitro and in vivo. Indeed, we also verified the expression of METTL3 and IGFBP3 in the atrial tissues of patients with atrial fibrillation (AF). Thus, METTL3 may regulate IGFBP3's expression and CFs activation via RNA epigenetic modifications, laying the foundation for a specific and novel therapeutic target in cardiac fibrosis.

Epigenetic control of LncRNA NEAT1 enables cardiac fibroblast pyroptosis and cardiac fibrosis.

Cardiac fibroblasts (CFs) drive extracellular matrix remodeling after inflammatory injury, leading to cardiac fibrosis and diastolic dysfunction. Recent studies described the role of epigenetics in cardiac fibrosis. Nevertheless, detailed reports on epigenetics regulating CFs pyroptosis and describing their implication in cardiac fibrosis are still unclear. Here, we found that DNMT3A reduces the expression of lncRNA Neat1 and promotes the NLRP3 axis leading to CFs pyroptosis, using cultured cells, animal models, and clinical samples to shed light on the underlying mechanism. We report that pyroptosis-related genes are increased explicitly in cardiac fibrosis tissue and LPS-treated CFs, while lncRNA Neat1 decreased. Mechanistically, we show that loss of DNMT3A or overexpression of lncRNA Neat1 in CFs after LPS treatment significantly enhances CFs pyroptosis and the production of pyroptosis-related markers in vitro. It has been demonstrated that DNMT3A can decrease lncRNA Neat1, promoting NLRP3 axis activation in CFs treated with LPS. In sum, this study is the first to identify that DNMT3A methylation decreases the expression of lncRNA Neat1 and promotes CFs pyroptosis and cardiac fibrosis, suggesting that DNMT3A and NEAT1 may function as an anti-fibrotic therapy target in cardiac fibrosis.

METTL3 boosts glycolysis and cardiac fibroblast proliferation by increasing AR methylation.

Dysregulated glycolysis has been noted in several pathological processes characterized by supporting cell proliferation. Nonetheless, the role of glycolysis reprogramming is not well appreciated in cardiac fibrosis which is accompanied by increased fibroblasts proliferation. In this study, we investigated the cause and consequence of glycolysis reprogramming in cardiac fibrosis, using clinical samples, animal models, and cultured cells. Herein, we report that methyltransferase-like 3 (METTL3) facilitates glycolysis and cardiac fibroblasts proliferation, leading to cardiac fibrosis. The augmentation of glycolysis, an essential event during cardiac fibroblasts proliferation, is dependent on an increased expression of METTL3. A knockdown of METTL3 suppressed glycolysis, and inhibited cardiac fibroblast proliferation and cardiac fibrosis. Mechanistically, METTL3 epigenetically repressed androgen receptor (AR) expression in an m6A-YTHDF2- dependent manner, by targeting the specific AR m6A site. AR could interact with the glycolysis marker HIF-1α, and down-regulation of AR activates HIF-1α signaling, resulting in enhanced glycolysis and cardiac fibroblast proliferation. In contrast, the overexpression of AR significantly reduced the HIF-1α axis, decreased expression of glycolytic enzymes HK3, inhibited glycolysis, and repressed cardiac fibroblasts proliferation. Notably, increased METTL3 and YTHDF2 levels, decreased AR expression, increased HIF-1α and Postn expression and augmented glycolysis, and increased cardiac fibrosis were detected in human atrial fibrillation heart tissues. Our results found a novel mechanism by which METTL3-catalyzed m6A modification in cardiac fibrosis, wherein it facilitated glycolysis and cardiac fibroblasts proliferation by increasing AR methylation in an m6A-YTHDF2- dependent manner and provided new insights strategies to intervene cardiac fibrosis.

MTHFR epigenetic derepression protects against diabetes cardiac fibrosis.

BACKGROUND: Diabetes cardiac fibrosis is associated with altered DNA methylation of fibrogenic genes; however, the underlying mechanisms remain unclear. OBJECTIVES: In this study, we investigate the critical role of DNA methylation aberration-associated suppression of MTHFR in diabetes cardiac fibrosis, and the protective effects of folate on diabetes cardiac fibrosis, using cultured cells, animal models, and clinical samples. METHODS AND RESULTS: Herein, we report that DNA methylation repression of MTHFR, critically involved in diabetes cardiac fibrosis, mediates the significant protective effects of folate in a mouse model of diabetes cardiac fibrosis induced by STZ. Heart MTHFR expression was markedly suppressed in diabetes cardiac fibrosis patients and mice, accompanied by increased DNMT3A and MTHFR promoter methylation. Knockdown of DNMT3A demethylated MTHFR promoter, recovered the MTHFR loss, and alleviated the diabetes cardiac fibrosis pathology and cardiac fibroblasts pyroptosis. Mechanistically, DNMT3A epigenetically repressed MTHFR expression via methylation of the promoter. Interestingly, folate supplementation can rescue the effect of MTHFR loss in diabetes cardiac fibrosis, suggesting that inactivation of MTHFR through epigenetics is a critical mediator of diabetes cardiac fibrosis. CONCLUSIONS: The current study identifies that MTHFR repression due to aberrant DNMT3A elevation and subsequent MTHFR promoter hypermethylation is likely an important epigenetic feature of diabetes cardiac fibrosis, and folate supplementation protects against diabetes cardiac fibrosis.

DNMT1-Induced miR-152-3p Suppression Facilitates Cardiac Fibroblast Activation in Cardiac Fibrosis.

Novel insights into epigenetic control of cardiac fibrosis are now emerging. Cardiac fibroblasts (CFs) activation into myofibroblasts and the production of extracellular matrix (ECM) is the key to cardiac fibrosis development, but the specific mechanism is not fully understood. In the present study, we found that DNMT1 hypermethylation reduces the expression of microRNA-152-3p (miR-152-3p) and promotes Wnt1/ß-catenin signaling pathway leading to CFs proliferation and activation. Cardiac fibrosis was produced by ISO, and the ISO was carried out according to the method described. CFs were harvested and cultured from SD neonatal rats and stimulated with TGF-ß1. Importantly, DNMT1 resulted in the inhibition of miR-152-3p in activated CFs and both DNMT1 and miR-152-3p altered Wnt/ß-catenin downstream protein levels. Over expression of DNMT1 and miR-152-3p inhibitors promotes proliferation of activating CFs. In addition, decreased methylation levels and over expression of miR-152-3p inhibited CFs proliferation. We determined that DNMT1 can methylate to miR-152-3p and demonstrated that expression of miR-152-3p inhibits CFs proliferation by inhibiting the Wnt1/ß-catenin pathway. Our results stand out together DNMT1 methylation regulates miR-152-3p to slow the progression of cardiac fibrosis by inhibiting the Wnt1/ß-catenin pathway.

Epigenetic aberrations of miR-369-5p and DNMT3A control Patched1 signal pathway in cardiac fibrosis.

Modulation of epigenetic marks has promised efficacy for treating fibrosis. Cardiac fibroblast is the primary source of activated myofibroblasts that produce extracellular matrix (ECM) in cardiac fibrosis, but the mechanisms underlying this process are incompletely understood. Here we show that microRNA-369-5p (miR-369-5p) through DNMT3A hypermethylation and suppression of the Patched1 pathway leads to fibroblast proliferation in cardiac fibrosis. Forty adult male Sprague-Dawley (SD) rats were randomly divided into two groups (sham and AAC group), cardiac fibrosis was produced by abdominal aortic constriction, and the operation of abdominal aortic constriction was carried out according to the method described. Cardiac fibroblasts (CFs) were harvested from SD neonate rats and cultured. Importantly, miR-369-5p bind directly to DNMT3A with high affinity. MiR-369-5p leads to inhibition of DNMT3A enzyme activity. Exogenous miR-369-5p in cells induces aberrant DNA methylation of the Patched1, resulting in hypermethylation of low to moderately methylated regions. Moreover, Overexpression of miR-369-5p in cardiac fibroblast cells inhibits proliferation. We identify DNMT3A as miR-369-5p target genes and demonstrate that inhibition of miR-369-5p expression augments cell proliferation by activating DNMT3A and suppression of the Patched1 pathway. Together, our results highlight miR-369-5p mediated DNMT3A epigenetic silencing of Patched1 as a mechanism of fibroblast proliferation in cardiac fibrosis.

Analgesic effects of melatonin on post-herpetic neuralgia.

OBJECTIVE: This study aims to explore the analgesic effects of melatonin on post-herpetic neuralgia and its possible mechanism. METHODS: A total of 48 PHN Wistar rats were divided into 4 groups randomly: Normal, PHN, PHN+MT and naloxone, 4P-PDOT or L-arginine+120 mg/kg MT (C). Heat pain latency was determined after MT injection for 20 min, 40 min, 80 min and 120 min respectively. The expression levels of δ receptor and MT2 receptor in different tissues of rats were detected by RT-PCR method. NO content was determined. RESULTS: Heat pain latency in PHN rats were lower than that of control group (P<0.05), MT could increase the heat pain latency with dose-dependent, while naloxone, 4P-PDOT and L-arginine could reverse the analgesic effect of MT (P<0.05). The expression levels of δ receptor and MT2 receptor in spinal cord, hypothalamus and hippocampus in PHN+MT (120 mg/kg, i. p.) group were significantly higher than that of PHN group (P<0.05). The NO levels in the brain and spinal cord tissues in PHN group were higher than that of PHN+MT (120 mg/kg) group (P<0.05). CONCLUSIONS: MT had significant analgesic effects in the treatment of PHN, and its mechanism was closely related with δopioid receptor, NO and MT2 receptor.