Mechanism of puerarin alleviating myocardial remodeling through NSUN2-mediated m5C methylation modification

Myocardial remodeling is a primary contributor to the development and progression of heart failure. Reversing this process is crucial for the prevention and treatment of heart failure. Puerarin (Pue), a traditional Chinese medicine, has been widely utilized in the management of cardiovascular diseases; however, its potential mechanisms for reversing myocardial remodeling remain unclear.

To investigate the effect of Pue in myocardial remodeling and the potential mechanism with m5C methylation.

A transverse aortic coarctation (TAC)-induced myocardial hypertrophy model was established in vivo, while angiotensin II (AngII)-induced cardiomyocyte hypertrophy was examined in vitro using H9c2 and NRCM cells. Various doses of Pue were administered to assess their effects. Myocardial remodeling was evaluated through echocardiography, H&E staining, WGA staining, Masson staining, PSR staining, and Western Blot (WB) analysis in mice, alongside mean cell area measurement and WB analysis in cultured cells. The m5C level was quantified using Dot Blot and immunohistochemistry (IHC). RNA sequencing, molecular docking, microscale thermophoresis (MST), transfection, immunofluorescence (IF), and WB were employed to confirm that Pue reverses myocardial remodeling via NSUN2-mediated m5C methylation modification in both in vitro and in vivo settings. The methylation modification was further validated using methylation immunoprecipitation (MeRIP-qPCR).

In both in vitro and in vivo experiments, we found that long-term administration of Pue can alleviate myocyte hypertrophy and fibrosis while improving both diastolic and systolic function in mice. Additionally, Pue was shown to upregulate the m5C modification level in cardiomyocytes. RNA sequencing, along with docking and MST assays, revealed that Pue upregulates and targets NSUN2. Furthermore, the protective effect of Pue was diminished by the knockdown of NSUN2 expression. Pue was found to inhibit the activation of NF-κB, and the knockdown of NSUN2 prolonged the half-life of NF-κB mRNA. The enrichment of m5C mRNA was confirmed through MeRIP-qPCR.

This study suggests that Pue enhances the m5C methylation of NF-κB mRNA by targeting NSUN2. The m5C methylation facilitates the degradation of NF-κB mRNA and suppresses NF-κB activation, thereby modulating the expression of factors related to myocardial remodeling. Consequently, this process alleviates myocardial remodeling and prevents the progression of heart failure, providing a novel perspective for the clinical treatment of heart failure.