Pathological cardiac hypertrophy is a major contributor of heart failure (HF), which seriously threatens human’s health world widely. Deregulation of m6A RNA methylation, and m6A methyltransferases and de-methyltransferases have been demonstrated to act essential roles in cardiac hypertrophy and HF. Here, we studied the potential roles and its underlying mechanisms of m6A Reader YTHDF proteins in HF. In this study, we constructed HF mouse model by transverse aortic constriction surgery. Primary cardiomyocytes were isolated and stimulated with isoproterenol (ISO) or phenylephrine (PHE) to induce myocardial hypertrophy. Through single-cell RNA-seq analysis, immunofluorescent staining, HE staining, Western blotting, and real time-PCR detections, we found that YTHDF2 mRNA and protein level, but not YTHDF1 or YTHDF3, was significantly increased during HF development. YTHDF2 overexpression could efficiently alleviate cardiac hypertrophy. Furthermore, through immunoprecipitation accompanied with mass spectrometry analysis, Gene Ontology (GO) analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, we found that ISO stimulation did not evidently affect YTHDF2-interacting proteins. However, ISO or PHE stimulation significantly increased YTHDF2 protein interacting with Myh7 (beta-myosin heavy chain) mRNA, an important cardiac hypertrophy marker, in an m6A-dependent manner. Knockdown of Myh7 or deletion of the YTH domain of YTHDF2 reversed the protective effects of YTHDF2 on cardiac hypertrophy. Finally, we found that ISO or PHE stimulation promoted YTHDF2 protein expression through enhancing Ythdf2 mRNA stability in an m6A-dependent manner in cardiomyocytes. Overall, our results indicate that the m6A Reader YTHDF2 suppresses cardiac hypertrophy via Myh7 mRNA decoy in an m6A-dependent manner. This study highlights the functional importance of YTHDF2-dependent cardiac m6A mRNA regulation during cardiac hypertrophy, and provides a novel mechanistic insight into the therapeutic mechanisms of YTHDF2.

中文翻译：

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YTHDF2通过调节Myh7 mRNA诱饵减轻心脏肥大

病理性心脏肥大是心力衰竭（HF）的主要诱因，严重威胁着人类的健康世界。m6A RNA 甲基化的失调以及 m6A 甲基转移酶和去甲基转移酶已被证明在心脏肥大和 HF 中起重要作用。在这里，我们研究了 m6A Reader YTHDF 蛋白在 HF 中的潜在作用及其潜在机制。在这项研究中，我们通过横向主动脉缩窄手术构建了 HF 小鼠模型。分离原代心肌细胞并用异丙肾上腺素 (ISO) 或去氧肾上腺素 (PHE) 刺激以诱导心肌肥大。通过单细胞 RNA-seq 分析、免疫荧光染色、HE 染色、Western blotting 和实时 PCR 检测，我们发现 YTHDF2 mRNA 和蛋白质水平，而不是 YTHDF1 或 YTHDF3，在 HF 发展期间显着增加。YTHDF2过表达可以有效缓解心脏肥大。此外，通过免疫沉淀伴随质谱分析、基因本体论 (GO) 分析和京都基因和基因组百科全书 (KEGG) 通路分析，我们发现 ISO 刺激对 YTHDF2 相互作用蛋白没有明显影响。然而，ISO 或 PHE 刺激以 m6A 依赖性方式显着增加了与 Myh7（β-肌球蛋白重链）mRNA（一种重要的心脏肥大标志物）相互作用的 YTHDF2 蛋白。Myh7 的敲除或 YTHDF2 的 YTH 结构域的缺失逆转了 YTHDF2 对心脏肥大的保护作用。最后，我们发现 ISO 或 PHE 刺激通过在心肌细胞中以 m6A 依赖性方式增强 Ythdf2 mRNA 稳定性来促进 YTHDF2 蛋白表达。总体而言，我们的结果表明 m6A Reader YTHDF2 以 m6A 依赖性方式通过 Myh7 mRNA 诱饵抑制心脏肥大。这项研究强调了在心脏肥大过程中 YTHDF2 依赖性心脏 m6A mRNA 调节的功能重要性，并提供了对 YTHDF2 治疗机制的新机制见解。