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 HF的重要危险因素。尽管有最重要的利益和迫切的需要，我们对 HF发展的机制仍然有限。以适应心脏收缩力的升高需求 在高血压下，心脏通过肥大生长而产生急性反应。这个曾经适应 反应可能失代偿并进展为HF。此更新应用程序的总体目标是描述 未折叠蛋白反应（UPR）在压力超负荷下HF中的作用。 累积的证据表明，UPR在压力超负荷和培养的心脏中被激活。 心肌细胞的生长刺激。在本项目的上一个供资期间， 功能研究已经证明剪接的XBP 1（sXBP 1），UPR的下游转录因子， 在血流动力学应激条件下对适应性心肌肥厚至关重要，这部分是由其靶点介导的 GFAT 1. sXBP 1是由UPR的三个信号转导子之一IRE 1 α在蛋白质折叠过程中产生的 应力在先前的资助研究过程中，IRE 1 α在翻译中的新作用，而不是sXBP 1控制的。 转录，被发现。这一作用可能为IRE 1 α的研究开辟了新的方向 以及心脏肥大和HF中的UPR。 通过无偏脉冲SILAC测定的初步结果表明，UPR蛋白富集 在细胞生长条件下。重要的是，只有IRE 1 α，而不是PERK或ATF 6，UPR的分支是必需的， 蛋白质合成和细胞生长。自sXBP 1恢复后，IRE 1 α的这种新作用不依赖于sXBP 1 不能挽救IRE 1 α沉默导致的生长缺陷。此外，年龄匹配的IRE 1 α条件性敲除（cKO） 与sXBP 1 cKO动物相比，小鼠表现出更严重的心肌病和HF。来自A的更多试验数据 蛋白质组学分析表明，IRE 1 α直接结合翻译起始复合物的组分。基于 这些发现提出了一个中心假设：IRE 1 α在翻译调控中发挥新的作用， 这是心脏在压力下产生适应性生长反应和对抗HF所必需的 超载。已进行核糖体分析以鉴定IRE 1 α的仅抑制靶点。IRE 1 α的作用 在翻译启动中，将通过测试翻译启动的组装和功能来描述 复杂.接下来，IRE 1 α靶向其靶标的5 '-和3'-UTR的机制将通过以下方法确定： 推导二级mRNA结构并在分子水平上验证。最后，IRE 1 α的效应子， 特别是膜蛋白，将评估它们对体内心脏肥大和HF的贡献。 阐明IRE 1 α和UPR在心脏重构和HF中的新作用将促进我们对心脏重构和HF的理解。 高血压性心脏病的病理生理学研究，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.