Cytoglobin: A stress-responsive hemoprotein modulating cardiomyocyte survival

描述（由申请人提供）：心力衰竭是美国心血管死亡的主要原因，通常是由于高血压或心肌梗塞 (MI) 导致的适应不良心脏重塑的结果。尽管过去十年我们在心力衰竭的治疗方面取得了重大进展，但与这种疾病相关的发病率和死亡率仍然很高。因此，识别预防或逆转适应不良心脏重塑过程的候选蛋白和信号通路是寻求新的治疗方法以提高心力衰竭患者的质量和寿命的主要目标。我们的实验室最近发现细胞球蛋白 (Cygb) 是一种应激反应性血红素蛋白，其水平在各种应激条件下（即缺氧、缺血和压力过载）下，在成人心脏中都会增加。 Cygb 在培养的心肌细胞中过度表达可以保护它们免受氧化应激，而 Cygb 的敲低会导致细胞凋亡增加。最初，我们假设 Cygb 通过所有血红素蛋白固有的抗氧化机制来保护心肌细胞；然而，我们现在有大量证据表明 Cygb 也直接参与调节肿瘤抑制蛋白 p53 的转录活性。 W 发现 Cygb 的转录水平与许多 p53 靶基因之间存在相互关系。染色质免疫沉淀实验证明 p53 和 Cygb 在经典 p53 靶启动子处共占据。使用重组蛋白，我们展示了两种蛋白质之间的直接蛋白质-蛋白质相互作用。重要的是，重组 Cygb 在体外重构转录测定中抑制 p53 转录活性。最后，我们开发了条件性 Cygb 敲除小鼠模型，并进行了初步研究，证明 Cygb 的急性心脏特异性缺失会导致扩张型心肌病。同样，与受伤的对照小鼠相比，出生时心脏特异性缺失 Cygb 的小鼠在缺血再灌注 (I/R) 损伤后会出现更明显的扩张型心肌病。我们的中心假设是 Cygb 通过抗氧化和 p53 依赖性机制促进心肌细胞存活。我们提出以下三个具体目标来检验这一假设： 具体目标 1：定义细胞珠蛋白在维持心肌细胞稳态中的作用。具体目标 2：定义细胞珠蛋白在适应不良心脏重塑过程中的作用。具体目标 3：确定细胞珠蛋白-p53 相互作用对心肌细胞存活的重要性。我们提出的研究的成功完成将深入了解调节心肌细胞存活的新信号通路，并可能为开发预防和/或逆转心力衰竭的新治疗方法提供机会。因此，拟议的 NIH R01 研究补助金申请与 NIH 和 NHBLI 的使命相关且一致。

DESCRIPTION (provided by applicant): Heart failure is the major cause of cardiovascular mortality in the US and often develops as a consequence of maladaptive cardiac remodeling due to hypertension or a myocardial infarction (MI). Although over the past decade we have made major advances in the treatment of heart failure, the morbidity and mortality associated with this disease remains high. Thus, the identification of candidate proteins and signaling pathways that prevent or reverse the process of maladaptive cardiac remodeling is a major goal in the pursuit of new treatment approaches for improving the quality and longevity of heart failure patients. Our laboratory recently identified cytoglobin (Cygb) as a stress-responsive hemoprotein and its levels increase in the adult heart under a variety of stress conditions (i.e. hypoxia, ischemia, and pressure-overload). Overexpression of Cygb in cultured myocytes protects them from oxidative stress, while knockdown of Cygb results in increased apoptotic cell death. Initially, we postulated that Cygb protects myocytes via anti-oxidative mechanisms inherent to all hemoproteins; however, we now have extensive evidence that Cygb is also directly involved in regulating the transcriptional activity of the tumor suppressor protein p53. W have found a reciprocal relationship between transcript levels of Cygb and a number of p53-target genes. Chromatin immunoprecipitation experiments demonstrate co-occupancy of p53 and Cygb at classic p53 target promoters. Using recombinant proteins we show a direct protein-protein interaction between the two proteins. Importantly, recombinant Cygb inhibits p53 transcriptional activity in vitro reconstituted transcriptional assays. Finally, we have developed conditional Cygb knockout mouse model and have preliminary studies demonstrating that acute cardiac-specific loss of Cygb leads to a dilated cardiomyopathy. Likewise, mice born with cardiac-specific deletion of Cygb develop a more pronounced dilated cardiomyopathy after ischemia-reperfusion (I/R) injury as compared to injured control mice. Our central hypothesis is that Cygb contributes to cardiomyocyte survival via both anti-oxidative and p53-dependent mechanisms. We propose the following three specific aims to test this hypothesis: Specific Aim 1: Define the role of cytoglobin in maintaining cardiomyocyte homeostasis. Specific Aim 2: Define the role of cytoglobin during maladaptive cardiac remodeling. Specific Aim 3: Define the significance of the cytoglobin-p53 interaction for cardiomyocyte survival. The successful completion of our proposed study will provide insight into a novel signaling pathway that regulates cardiomyocyte survival and may provide opportunities for the development of novel therapeutic approaches to prevent and/or reverse heart failure. Thus, the proposed NIH R01 Research Grant Application is relevant to and in keeping with the missions of both the NIH and NHBLI.