Xie Yanan, Jiao Wei, Wang Hongchao, Kang Shaowei, Hao Jie, Liu Jinming, Li Yongjun, Guo Bingyan
Department of Cardiology, The Second Hospital of Hebei Medical University.
Int Heart J. 2025;66(4):615-627. doi: 10.1536/ihj.24-683.
The aim of this study was to investigate the mechanism of m6A methylation in pathological myocardial hypertrophy (PMH), focusing on whether the methyltransferase METTL3 regulates the expression and nuclear translocation of the transcription factor EB (TFEB), thereby affecting autophagic activity and exacerbating the development of myocardial hypertrophy.An in vivo PMH model was established in mice via transverse aortic constriction (TAC), and an in vitro hypertrophy model was established using H9C2 cells stimulated with angiotensin II (AngII). HE staining, Western blotting, qRT-PCR, immunofluorescence, and dual-color autophagy flux analyses were employed to detect the expression of autophagy-related proteins (LC3, Beclin-1, P62, ATG5) and apoptosis levels. Changes in TFEB and key m6A-related enzymes (METTL3, ALKBH5, heterogeneous nuclear ribonucleoprotein D [HNRNPD]) were examined, and gene overexpression or knockdown experiments were performed to clarify their roles in regulating autophagy and myocardial hypertrophy. Finally, m6A MeRIP-qPCR and RIP-qPCR were conducted to explore the effect of METTL3 on m6A modification and the stability of TFEB transcripts, verifying the interplay between METTL3 and TFEB and its impact on autophagy.In both the in vivo and in vitro hypertrophy models, autophagy was significantly impaired and apoptosis was elevated, while TFEB mRNA and protein expression and its nuclear localization were clearly reduced. At the same time, global m6A methylation was markedly increased, accompanied by upregulation of METTL3 and HNRNPD, as well as downregulation of ALKBH5. Functional assays indicated that METTL3 overexpression further inhibited autophagy-related protein expression and autophagic flux, whereas METTL3 knockdown partially restored autophagy. Mechanistic studies revealed that METTL3 modulates TFEB pre-mRNA stability by influencing the binding efficiencies of ALKBH5 and HNRNPD, resulting in decreased TFEB expression. Conversely, overexpression of TFEB could partly counteract the autophagy impairment caused by METTL3 overexpression and reciprocally regulate the expression of METTL3, ALKBH5, and HNRNPD.METTL3 mediates the inhibition of TFEB via m6A modification, thereby impairing autophagy and aggravating myocardial hypertrophy. These findings suggest that the m6A-TFEB axis may serve as a novel therapeutic target for preventing and treating myocardial hypertrophy and heart failure, offering new insights into the intervention of cardiac remodeling-related diseases.
本研究旨在探讨N6-甲基腺苷（m6A）甲基化在病理性心肌肥大（PMH）中的作用机制，重点关注甲基转移酶METTL3是否调节转录因子EB（TFEB）的表达和核转位，从而影响自噬活性并加剧心肌肥大的发展。通过横向主动脉缩窄（TAC）在小鼠体内建立PMH模型，并使用血管紧张素II（AngII）刺激的H9C2细胞建立体外肥大模型。采用苏木精-伊红（HE）染色、蛋白质免疫印迹法、实时定量聚合酶链反应（qRT-PCR）、免疫荧光和双色自噬通量分析来检测自噬相关蛋白（微管相关蛋白轻链3[LC3]、Beclin-1、P62、自噬相关蛋白5[ATG5]）的表达和凋亡水平。检测TFEB和关键的m6A相关酶（METTL3、alkB同源蛋白5[ALKBH5]、不均一核核糖核蛋白D[HNRNPD]）的变化，并进行基因过表达或敲低实验以阐明它们在调节自噬和心肌肥大中的作用。最后，进行m6A甲基化RNA免疫沉淀定量PCR（m6A MeRIP-qPCR）和RNA免疫沉淀定量PCR（RIP-qPCR）以探讨METTL3对m6A修饰和TFEB转录本稳定性的影响，验证METTL3与TFEB之间的相互作用及其对自噬的影响。
在体内和体外肥大模型中，自噬均显著受损，凋亡增加，而TFEB的mRNA和蛋白表达及其核定位明显降低。同时，整体m6A甲基化显著增加，伴随着METTL3和HNRNPD的上调以及ALKBH5的下调。功能实验表明，METTL3过表达进一步抑制自噬相关蛋白表达和自噬通量，而METTL3敲低部分恢复了自噬。机制研究表明，METTL3通过影响ALKBH5和HNRNPD的结合效率来调节TFEB前体mRNA的稳定性，导致TFEB表达降低。相反，TFEB过表达可部分抵消METTL3过表达引起的自噬损伤，并相互调节METTL3、ALKBH5和HNRNPD的表达。
METTL3通过m6A修饰介导对TFEB的抑制，从而损害自噬并加重心肌肥大。这些发现表明，m6A-TFEB轴可能成为预防和治疗心肌肥大及心力衰竭的新治疗靶点，为心脏重塑相关疾病的干预提供了新的见解。
