Translational Control of Cardiac Fibrosis

项目摘要： 心血管疾病（CVD）是全球发病率和死亡率的主要原因。心力衰竭 (HF)心血管疾病的一种主要形式，由心肌细胞（CM）肥大和凋亡引起， 心脏成纤维细胞（CF）活化和增殖。虽然TGF-β-IL-11轴诱导蛋白质合成 已经观察到促纤维化基因，促进促纤维化mRNA翻译的机制 在CF中尚未确定。因此，填补这一空白， 心脏纤维化的可靶向的翻译控制决定子是极其重要的。 氨酰-tRNA合成酶（ARS）催化氨基酸与同源tRNA的连接。在 在哺乳动物中，谷氨酰-脯氨酰-tRNA合成酶（EPRS）催化谷氨酸（E）的连接， 脯氨酸（P），与它们的同源tRNA结合用于蛋白质合成。通过筛选TGF-b诱导的ARS， 活化的人CF、CHD中具有SNP的人ARS、与ISO诱导的 GWAS引起的心肌病，以及ISO诱导的小鼠心脏衰竭中的ARS，我们已经确定EPRS为 参与各种心脏病理的关键ARS。在这个项目中，我们发现EPRS是一个 多个促纤维化刺激下游的集成节点。我们发现转录激活因子 5作为EPRS的上游转录调节因子。Halofuginone（Halo），a Chinese Herb 药物衍生化合物，是脯氨酰-tRNA合成酶特异性抑制剂。低剂量光环， 以及小鼠基因组中EPRS的一个等位基因的基因敲除，减少了心脏肥大， 在多种HF小鼠模型中的纤维化。我们在Halo中采用了RNA测序和多聚核糖体分析测序， 处理的成纤维细胞，以确定转录组和翻译组水平上的基因表达变化， EPRS抑制。我们发现了新的富含脯氨酸密码子（PRR）的基因，除了胶原蛋白， EPRS在转录后水平上调，可能在心脏纤维化中起关键作用。 EPRS的失活促进PRR mRNA的翻译抑制与增强的mRNA偶联 腐烂这种效应需要eIF 5A的失活，eIF 5A是一种用于解码富含Pro的密码子的延伸因子。 我们的中心假设是：MI应激诱导的EPRS通过增加Pro-tRNAPro促进心脏纤维化 池，并增强CF中促纤维化PRR mRNA的稳定性和翻译激活。我们将 通过追求三个目标来检验这个假设。目标1。确定是否在全球范围内和CF中下调，以及 EPRS的药理学抑制预防或逆转HF模型中的心脏纤维化。目标二。确定 EPRS和上游调节因子的新型优先下游PRR促纤维化mRNA靶点 EPRS的路径。目标3：确定eIF 5A介导的翻译控制和mRNA的机制 PRR基因的衰变。该项目将通过以下方式促进新型治疗方法的发展： 抑制翻译因子和鉴定新的药理学靶点。

Project Summary: Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide. Heart failure (HF), a major form of CVD, results from cardiomyocyte (CM) hypertrophy and apoptosis, combined with cardiac fibroblast (CF) activation and proliferation. Although TGF-b-IL-11 axis induced protein synthesis of pro-fibrotic genes have been observed, the mechanisms that promote pro-fibrotic mRNA translation in CF have not been identified. Therefore, filling this gap and identifying the pharmacologically targetable translational control determinants of cardiac fibrosis is of paramount importance. Aminoacyl-tRNA synthetases (ARSs) catalyze the ligation of amino acids to cognate tRNAs. In mammals, glutamyl-prolyl-tRNA synthetase (EPRS) catalyzes the attachment of glutamic acid (E) and proline (P), to their cognate tRNAs for protein synthesis. By screening of ARSs induced in TGF-b- activated human CFs, human ARSs with SNPs in CHD, mouse ARSs associated with ISO-induced cardiomyopathy by GWAS, and ISO-induced ARSs in mouse failing hearts, we have identified EPRS as the key ARS involved in various cardiac pathologies. In this project, we uncovered EPRS as an integrated node downstream of multiple pro-fibrotic stimuli. We found that activating transcription factor 5 acts as an upstream transcriptional regulator of EPRS. Halofuginone (Halo), a Chinese herbal medicine-derived chemical compound, is a prolyl-tRNA synthetase-specific inhibitor. Low-dose Halo, as well as genetic knockout of one allele of EPRS in the mouse genome, reduces cardiac hypertrophy and fibrosis in multiple HF mouse models. We employed RNA-Seq and polysome profiling-Seq in Halo- treated fibroblasts to define gene expression changes at the transcriptomic and translatomic level upon EPRS inhibition. We identified novel proline codon rich (PRR) genes in addition to collagens, which are upregulated by EPRS at the posttranscriptional level and may play critical roles in cardiac fibrosis. Inactivation of EPRS promotes translational repression of PRR mRNA coupled with enhanced mRNA decay. This effect requires inactivation of eIF5A, an elongation factor for decoding Pro-rich codons. Our central hypothesis is: MI stress-induced EPRS promote cardiac fibrosis via increased Pro-tRNAPro pool, and enhanced stabilization and translational activation of pro-fibrotic PRR mRNAs in CFs. We will test this hypothesis by pursuing 3 aims. Aim 1. Determine if downregulation, globally and in CFs, and pharmacological inhibition of EPRS prevent or reverse cardiac fibrosis in HF models. Aim 2. Determine novel preferential downstream PRR pro-fibrotic mRNA targets of EPRS and upstream regulatory pathway of EPRS. Aim 3. Determine mechanisms of eIF5A-mediated translational control and mRNA decay of PRR genes. This project will promote the development of novel therapeutic approaches by inhibiting translation factors, and identification of novel pharmacological targets.