RNA Binding Patterns of Ischemic Heart Failure Prognosis Indicator Eukaryotic Translation Initiation Factor 3m Reveal its Unique Role in Regulation of Nitrative Damage after Myocardial Ischemia-Reperfusion [ClIP-Seq]

RNA undergoes various modifications in a manner similar to DNA, thereby opening up the research field of epitranscriptomics. Regulators of mRNA modifications play a role in many molecular processes, biological functions, and disease prognosis. Differential expression profile of RNA modification regulators from HFrEF patients’ hearts remains deficient at present, let alone their affections on the prognosis. A risk score model based on two modifications-related modulators (eIF3m and Nudt12) was built and screened out with a LASSO regression analysis. Translation dysregulation upon eIF3m knock down in cardiomyocytes was examined by combined high-throughput methods of transcriptome sequencing, ribosome profiling, proteome research, and Clip analyses. Metabolic assays and flux analysis were used to characterize the effects of eIF3m and its downstream mediator in metabolic reprogramming with high-level exogenous TNF-α to mimic a highly oxidative inflammatory injury. eIF3m deficiency leads to decoupling of the major steps in translation initiation and elongation. It stabilizes eIF3m-f-h subcomplex through proteasome dependent-polyubiquitination, and controls translational response of specific mRNAs. eIF3m directly binds to Mt2 5’ leader to upregulate its translation in NRCMs and H9c2 cells. eIF3m knockdown induced expression of genes involved in proinflammatory and oxidative stress, resulting in 3-NT modification to a key glycolysis enzyme Pfkfb3 when co-cultured with exogenous TNF-α. CM-specific eIF3m deletion deteriorates cardiac function and increases infarction size after I/R operation, whereas CM-specific eIF3m overexpression reverses the phenotype. Additionally, eIF3m overexpression inhibits whereas RNAi-mediated Mt2 knockdown activates NF-κB signaling and aggravates nitrative damage. Metabolic analyses highlighted the role for eIF3m in promoting glycolysis and biosynthesis required for functional recovery of the myocardium. Our study demonstrates that the prognosis indicator eIF3m alleviates oxidative inflammatory injury and promotes glycolytic reprograming mainly by binding to Mt2 mRNA. Therapeutic strategies enhancing eIF3m level may possess therapeutic potential against I/R injury.RNA与DNA类似，可发生多种修饰，由此开创了表观转录组学（epitranscriptomics）的研究领域。mRNA修饰调控因子参与诸多分子过程、生物学功能及疾病预后调控。目前针对射血分数降低的心力衰竭（heart failure with reduced ejection fraction, HFrEF）患者心脏组织中RNA修饰调控因子的差异表达谱研究仍较为匮乏，其对疾病预后的影响更是鲜有报道。本研究通过套索（LASSO）回归分析，构建并筛选得到一个基于两种修饰相关调控因子（eIF3m与Nudt12）的风险评分模型。研究者采用转录组测序、核糖体谱分析、蛋白质组研究及交联免疫沉淀（CLIP）分析等高通量联合实验方法，检测了心肌细胞中敲低eIF3m后引发的翻译失调情况。本研究还借助代谢实验与通量分析，在以高水平外源性肿瘤坏死因子α（TNF-α）模拟重度氧化炎性损伤的模型中，表征了eIF3m及其下游介导因子在代谢重编程中的作用。研究发现，eIF3m缺失会导致翻译起始与延伸的关键步骤发生解偶联；其可通过蛋白酶体依赖的多泛素化过程稳定eIF3m-f-h亚复合物，并调控特定mRNA的翻译响应。eIF3m可直接结合Mt2的5'非翻译区，上调其在新生大鼠心肌细胞（NRCMs）与H9c2细胞中的翻译水平。在外源性TNF-α共培养条件下，敲低eIF3m会促进促炎及氧化应激相关基因的表达上调，导致关键糖酵解酶Pfkfb3发生3-硝基酪氨酸（3-NT）修饰。心肌细胞特异性敲除eIF3m会加重缺血再灌注（I/R）手术后的心脏功能损伤并增大梗死面积，而心肌细胞特异性过表达eIF3m则可逆转该表型。此外，过表达eIF3m会抑制核因子κB（NF-κB）信号通路，而RNA干扰介导的Mt2敲低则会激活该通路并加重硝化损伤。代谢分析揭示了eIF3m在促进心肌功能恢复所需的糖酵解与生物合成过程中的关键作用。本研究证实，预后标志物eIF3m主要通过结合Mt2 mRNA，缓解氧化炎性损伤并促进糖酵解重编程。提升eIF3m水平的治疗策略或许有望成为对抗缺血再灌注损伤的潜在治疗手段。