Role of the unfolded protein response in pathological cardiac remodeling

项目摘要 心力衰竭(HF)是全球发病率和死亡率的主要原因。高血压是最常见的 心衰的重要危险因素。尽管利益至上，需求迫切，但我们对 心力衰竭的发生机制仍然有限。以适应对心脏收缩能力的更高要求 在高血压的情况下，心脏会通过肥大的生长产生剧烈的反应。这曾经是自适应的 反应可能失代偿并进展为心衰。此续订申请的总体目标是描述 未折叠蛋白反应(UPR)在压力超负荷下心衰中的作用。 累积证据表明，在压力超负荷和培养条件下，UPR在心脏中被激活 心肌细胞通过生长刺激。在本项目的前一个资金期内，损益均为 功能研究表明，剪接的XBP1(SXBP1)是UPR的下游转录因子， 在血流动力学应激下的适应性心肌肥厚中起关键作用，这部分是由其靶点介导的。 GFAT1.SXBP1是由UPR的三个信号转导分子之一的Ire 1α在蛋白质折叠下产生的 压力。在之前的资助研究过程中，IRE1α在翻译中的一个新角色，而不是sXBP1控制的 抄本，被发现了。这一作用不是由sXBP1介导的，可能会为IRE1α开辟新的研究方向 心肌肥厚和心力衰竭时的UPR。 通过无偏脉冲SILAC分析的初步结果表明，UPR蛋白被浓缩 在细胞生长条件下。重要的是，只需要普遍定期审议的IRE1α，而不是PERK或ATF6分支 蛋白质合成和细胞生长。自从sXBP1恢复以来，IRE1α的这一新作用独立于sXBP1 不能从IRE1α沉默中挽救生长缺陷。此外，年龄匹配的Ire 1α条件基因敲除(CKO) 与sXBP1 CKO动物相比，小鼠表现出更严重的心肌病和心力衰竭。进一步的试点数据来自 蛋白质组学分析表明，IRE1α直接与翻译起始复合体的组成部分结合。基于 这些发现提出了一个中心假设：IRE1α在翻译调控中发挥了新的作用， 这对于心脏在压力下启动适应性生长反应和对抗HF是必不可少的 超载。核糖体分析已经被用来识别IRE1α的仅翻译靶标。Ire_1α的作用 将通过测试翻译起始的组装和功能来描述翻译起始 很复杂。接下来，IRE1α在其靶标的5‘-和3’-UTRs上的靶向机制将通过以下方式确定 从分子水平推导出二级结构并进行验证。最后，IRE1α的效应器， 特别是膜蛋白，将评估它们在体内对心肌肥厚和心衰的贡献。 阐明IRE1、α和UPR在心脏重构和心力衰竭中的新作用将促进我们的理解 研究高血压心脏病的病理生理学，并为新的、更有效的治疗设计铺平道路。

Project Summary Heart failure (HF) is a leading cause of morbidity and mortality worldwide. Hypertension is one of the most important risk factors of HF. Despite paramount interests and urgent needs, our understanding of the mechanisms of HF development remains limited. To accommodate the elevated demand of cardiac contractility under high blood pressure, the heart mounts an acute reaction through hypertrophic growth. This once adaptive response may decompensate and progress into HF. The overall goal of this renewal application is to delineate the role of the unfolded protein response (UPR) in HF under pressure overload. Cumulative evidence shows that the UPR is activated in the heart under pressure overload and in cultured cardiomyocytes by growth stimulation. During the previous funding period of this project, both gain- and loss-of- function studies have demonstrated that spliced XBP1 (sXBP1), a downstream transcriptional factor of the UPR, is critical for adaptive cardiac hypertrophy under hemodynamic stress, which is partly mediated by its target GFAT1. sXBP1 is produced by IRE1α, one of the three signaling transducers of the UPR, under protein-folding stress. In the course of the previous funding studies, a novel role of IRE1α in translation, not sXBP1-controlled transcription, was uncovered. This action, not mediated by sXBP1, may open a new research direction of IRE1α and the UPR in cardiac hypertrophy and HF. Preliminary results through an unbiased pulse SILAC assay demonstrate that UPR proteins are enriched under cell growth conditions. Importantly, only the IRE1α, not PERK or ATF6, branch of the UPR is required for protein synthesis and cell growth. This new action of IRE1α is independent of sXBP1 since sXBP1 restoration does not rescue growth defect from IRE1α silencing. Moreover, age-matched IRE1α conditional knockout (cKO) mice present more severe cardiomyopathy and HF compared to sXBP1 cKO animals. Further pilot data from a proteomic assay suggest that IRE1α directly binds components of the translation initiation complex. Based on these findings, a central hypothesis has been proposed: IRE1α exerts a new role in translational regulation, which is essential for the heart to mount an adaptive growth response and antagonize HF under pressure overload. Ribosome profiling has been conducted to identify translation-only targets of IRE1α. The role of IRE1α in translation initiation will be delineated by testing the assembly and functionality of the translation initiation complex. Next, the mechanism of targeting of IRE1α on 5’- and 3’-UTRs of its targets will be determined by deducing secondary mRNA structures and validation at the molecular level. Finally, effectors of IRE1α, particularly membrane proteins, will be evaluated for their contributions to cardiac hypertrophy and HF in vivo. Elucidation of the novel role of IRE1α and the UPR in cardiac remodeling and HF will advance our understanding of the pathophysiology of hypertensive heart disease and pave a way for novel, more effective therapeutic design.