Translational Control of Oxidative Stress in Myocardial Infarction

描述(由申请人提供)：已知的缺血再灌注(I/R)可引起心肌内活性氧的增加。尽管高水平的氧化剂可以造成损伤，但低剂量的氧化剂可以产生保护作用，例如在短暂I/R循环诱导的预适应期间。我们最近的数据表明，抗氧化和解毒基因的诱导在心肌细胞氧化剂的基因表达网络中占据主导地位。控制这些基因表达的主开关是转录因子Nrf2。我们发现，2~4个循环的5min缺血和5min再灌流可引起心肌中Nrf2蛋白的升高。对于分离的心肌细胞，氧化剂导致Nrf2蛋白翻译的快速启动。压力通常会导致蛋白质翻译的全球性减少。最近的研究表明，在mRNA的5‘非翻译区含有内部核糖体进入位点(IRES)的几个基因可以逃避一般的翻译控制，进行应激诱导的蛋白质翻译。一组IRES反式作用因子(ITAFs)似乎在招募核糖体来启动这些基因的翻译方面起着关键作用。当心肌或心肌细胞遇到氧化应激时，人们对哪些蛋白质以及它们是如何翻译的知之甚少。我们计划利用基因组和蛋白质组学技术来验证这一假设，即“短暂的I/R循环通过IRES介导的翻译导致心肌中NRF-2蛋白选择性增加”。具体目的包括1)测试Nrf2蛋白翻译通过短暂的I/R循环发生在心肌中并介导预适应诱导的心脏保护；2)测试5‘UTR中缺乏明确定义的Kozak序列和形成稳定的“茎和环”二级结构使一组基因能够经历I/R诱导的蛋白质翻译；3)测试Nrf2 mRNA包含允许在短暂I/R循环或低剂量氧化剂的循环后在心肌细胞中快速蛋白质翻译的IRES；4)检测心肌细胞是否表达一组独特的ITAFs来调节Nrf2蛋白的翻译，以响应I/R。应激诱导的蛋白翻译领域还处于起步阶段。尽管Nrf2在多种细胞类型中起着重要的细胞保护作用，但Nrf2在心肌细胞中的功能和调控还没有得到很好的研究。研究氧化应激下蛋白质翻译的机制成为理解适应性反应向心力衰竭转变的必要任务。公共卫生相关性：这项拨款建议研究Nrf2蛋白在心肌细胞和氧化应激后心肌中的翻译机制。

DESCRIPTION (provided by applicant): Ischemic reperfusion (I/R) is known to trigger an increase of reactive oxygen species in the myocardium. Although high levels of oxidants can cause damages, low doses of oxidant elicit a protective effect, such as during preconditioning induced by cycles of brief I/R. Our recent data suggest that induction of antioxidant and detoxification genes dominates the gene expression network of oxidants in cardiomyocytes. A master switch controlling the expression of these genes is the transcription factor Nrf2. We found that 2 to 4 cycles of 5 min ischemia and 5 min reperfusion cause elevation of Nrf2 protein in the myocardium. With isolated cardiomyocytes, oxidants cause rapid onset of Nrf2 protein translation. Stress generally causes global reduction of protein translation. Recent studies suggest that several genes containing an Internal Ribosomal Entry Site (IRES) in the 5' Untranslated Region (5'UTR) of mRNA can escape the general translational control and undergo stress induced protein translation. A group of IRES Trans-Acting Factors (ITAFs) appears to be critical in recruiting the ribosomes to initiate the translation of these genes. Little is known about which proteins and how they are translated when the myocardium or cardiomyocytes encounter oxidative stress. We plan to take advantage of genomic and proteomic technologies to test the hypothesis that "cycles of brief I/R cause selective increase of Nrf-2 protein in the myocardium through IRES mediated translation". The Specific Aims include 1) Test that Nrf2 protein translation occurs in the myocardium by cycles of brief I/R and mediates preconditioning induced cardiac protection; 2) Test that lack of well defined Kozak sequence and formation of stable "stems and loops" secondary structure in 5'UTR enable a battery of genes to undergo I/R induced protein translation; 3) Test that Nrf2 mRNA contains an IRES that allows rapid protein translation in cardiomyocytes following cycles of brief I/R or low dose of oxidants; 4) Test that cardiomyocytes express a unique set of ITAFs to regulate Nrf2 protein translation in response to I/R. The field of stress induced protein translation is in its stage of infancy. Although Nrf2 plays a critical role in cytoprotection among various cell types, the function and regulation of Nrf2 have not been well studied in cardiomyocytes. Studying the mechanism of protein translation under oxidative stress becomes a necessary task in understanding the transition to heart failure from adaptive response. PUBLIC HEALTH RELEVANCE: This grant proposes to study the mechanism of Nrf2 protein translation in cardiomyocytes and in the myocardium following oxidative stress.