m6A methyltransferase METTL3 participated in sympathetic neural remodeling post-MI via the TRAF6/NF-κB pathway and ROS production.

OBJECTIVE: Sudden cardiac death caused by ventricular arrhythmias (VAs) is the main cause of high mortality in patients with myocardial infarction (MI). Sympathetic neural remodeling caused by inflammation after MI is closely associated with the occurrence of VAs. METTL3, the earliest identified m6A methyltransferase, is critical in mediating inflammatory responses. Our aim was to investigate whether the m6A methyltransferase METTL3 was involved in sympathetic remodeling post-MI and its specific mechanism. METHODS AND RESULTS: A rat MI model was established via left coronary artery ligation. The expression of METTL3, TRAF6, NOX2, and NF-κB increased at 3 days and remained elevated at 7 days after MI, as determined via Western blotting. METTL3 was primarily present in macrophages, as determined via immunofluorescence. Intramyocardial injection of lentivirus carrying METTL3-shRNA inhibited METTL3 expression in vivo. Methylated immunoprecipitation-qPCR determined the METTL3 knockdown inhibited the m6A level of TRAF6 mRNA 3'-UTR. The co-immunoprecipitation experiment proved that METTL3 combines with TRAF6. Western blotting showed that silencing METTL3 inhibited TRAF6 level, NF-κB activation, and ROS production; decreased cytokine release (TNF-α and IL-1ß); and downregulated nerve growth factor expression. Finally, METTL3 knockdown reduced sympathetic remodeling after MI, as determined via immunofluorescence assays of tyrosine hydroxylase and growth-associated protein 43. Programmed electrical stimulation, renal sympathetic nerve activity recording, and haemodynamic measurements showed that METTL3 inhibition decreased sympathetic activity and improved cardiac function. CONCLUSIONS: Downregulation of METTL3 expression attenuated the excessive sympathetic neural remodeling induced by MI, further reducing the incidence of VAs and improving cardiac function. This was partly associated with the inhibition of the TRAF6/NF-κB pathway and ROS production.

Effect of TLR4/MyD88/NF-kB axis in paraventricular nucleus on ventricular arrhythmias induced by sympathetic hyperexcitation in post-myocardial infarction rats.

Sympathetic activation after myocardial infarction (MI) leads to ventricular arrhythmias (VAs), which can result in sudden cardiac death (SCD). The toll-like receptor 4 (TLR4)/myeloid differentiation primary response 88 (MyD88)/nuclear factor-kappa B (NF-kB) axis within the hypothalamic paraventricular nucleus (PVN), a cardiac-neural sympathetic nerve centre, plays an important role in causing VAs. An MI rat model and a PVN-TLR4 knockdown model were constructed. The levels of protein were detected by Western blotting and immunofluorescence, and localizations were visualized by multiple immunofluorescence staining. Central and peripheral sympathetic activation was visualized by immunohistochemistry for c-fos protein, renal sympathetic nerve activity (RSNA) measurement, heart rate variability (HRV) analysis and norepinephrine (NE) level detection in serum and myocardial tissue measured by ELISA. The arrhythmia scores were measured by programmed electrical stimulation (PES), and cardiac function was detected by the pressure-volume loop (P-V loop). The levels of TLR4 and MyD88 and the nuclear translocation of NF-kB within the PVN were increased after MI, while sympathetic activation and arrhythmia scores were increased and cardiac function was decreased. However, inhibition of TLR4 significantly reversed these conditions. PVN-mediated sympathetic activation via the TLR4/MyD88/NF-kB axis ultimately leads to the development of VAs after MI.

New insights into the central sympathetic hyperactivity post-myocardial infarction: Roles of METTL3-mediated m6 A methylation.

Ventricular arrhythmias (VAs) triggers by sympathetic nerve hyperactivity contribute to sudden cardiac death in myocardial infarction (MI) patients. Microglia-mediated inflammation in the paraventricular nucleus (PVN) is involved in sympathetic hyperactivity after MI. N6-methyladenosine (m6 A), the most prevalent mRNA and epigenetic modification, is critical for mediating cell inflammation. We aimed to explore whether METTL3-mediated m6 A modification is involved in microglia-mediated sympathetic hyperactivity after MI in the PVN. MI model was established by left coronary artery ligation. METTL3-mediated m6 A modification was markedly increased in the PVN at 3 days after MI, and METTL3 was primarily located in microglia by immunofluorescence. RNA-seq, MeRIP-seq, MeRIP-qPCR, immunohistochemistry, ELISA, heart rate variability measurements, renal sympathetic nerve activity recording and programmed electrical stimulation confirmed that the elevated toll-like receptor 4 (TLR4) expression by m6 A modification on TLR4 mRNA 3'-UTR region combined with activated NF-κB signalling led to the overwhelming production of pro-inflammatory cytokines IL-1ß and TNF-α in the PVN, thus inducing the sympathetic hyperactivity and increasing the incidence of VAs post-MI. Targeting METTL3 attenuated the inflammatory response and sympathetic hyperactivity and reduced the incidence of VAs post-MI.

EphrinB2-RhoA upregulation attenuates sympathetic hyperinnervation and decreases the incidence of ventricular arrhythmia after myocardial infarction.

BACKGROUND: Cardiac sympathetic hyperinnervation after myocardial infarction (MI) is associated with a high incidence of lethal arrhythmia. Erythropoietin-producing hepatoma interactor B2 (EphrinB2), a diffusible axonal chemorepellent that can induce growth cone collapse and axon repulsion of several neuronal populations, is crucial in neurodevelopment during disease development and progression. However, whether EphrinB2 could inhibit cardiac sympathetic hyperinnervation after MI remains unclear. METHODS AND RESULTS: A rat model of MI was developed by left anterior descending coronary artery ligation. EphrinB2 expression was markedly increased in the infarcted border at 3 days after MI. Downregulation of EphrinB2 by intramyocardial injection of lentivirus carrying EphrinB2-shRNA significantly increased sympathetic hyperinnervation along with downregulated RhoA expression. In contrast, injection of EphrinB2-overexpressing lentivirus markedly upregulated EphrinB2, concomitant with inhibition of sympathetic sprouting and upregulated RhoA expression, accompanied by decreased incidence of ventricular arrhythmias (VAs). However, co-administering EphrinB2-overexpressing lentivirus and Fasudil (Rho kinase inhibitor) nearly abolished the inhibition of nerve sprouting effect. Additionally, EphrinB2 expression did not affect nerve growth factor level in the infarcted heart. CONCLUSIONS: Overexpression of EphrinB2 may ameliorate MI-induced sympathetic hyperinnervation and further reduce the incidence of VAs, at least in part by activating RhoA-mediated axonal retraction.

[Expression of neuropilin 2 in human colorectal carcinoma and its correlation with lymphangiogenesis and lymphatic metastasis of tumor].

OBJECTIVE: To explore the relationship between neuropilin 2 (NRP-2) and lymphangiogenesis and lymphatic metastasis of human colorectal carcinoma (CRC), as well as the expression of NRP-2 in CRC tissues. METHODS: A total of 55 cases of CRC, adjacent and normal tissues of surgical resection were randomly selected at our hospital from March 2010 to January 2012. All pathological findings were confirmed by histopathology. The expression of NRP-2 was detected with reverse transcription (RT)-PCR and immunohistochemistry in parenchymatous and surrounding malignant tissues. Then lymphangiogenesis was marked with D2-40 monoclonal antibody and microlymphatic density (MLD) counted. RESULTS: Significant differences of MLD existed between those of tumor region (39 ± 19) and tumor margin (53 ± 26, P < 0.01). Both the number and shape of lymphangiogenesis were different between the parenchymatous and surrounding tissues. The expression of NRP-2 had a positive correlation with MLD both at the protein level (r = 0.325, P < 0.05) and at the gene level (r = 0.545, P = 0.000). And it was also correlated with the differentiation degree, infiltrative degree, lymphovascular invasion, lymph node metastasis and Dukes tumor staging (all P < 0.05). CONCLUSION: The expression of NRP-2 may regulate lymphangiogenesis and it may play an important role in the incidence and development of CRC.