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死（MI）引起的适应不良心脏重塑的结果。尽管在过去的十年中，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，鉴定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用于改善心力衰竭患者的质量和寿命的新治疗方法的主要目标。我们的实验室最近确定了细胞珠蛋白（Cygb）作为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，缺血和压力超负荷）的成人心脏增加。Cygb在培养的心肌细胞中的过表达保护它们免受氧化应激，而Cygb的敲低导致凋亡性细胞死亡增加。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。我们已经发现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实了p53和Cygb在经典p53靶启动子处的共占据。使用重组蛋白，我们显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在体外重建的转录测定中抑制p53转录活性。最后，我们建立了条件性Cygb基因敲除小鼠模型，并进行了初步研究，证明急性心脏特异性Cygb缺失导致扩张型心肌病。同样地，与受伤的对照小鼠相比，出生时具有心脏特异性Cygb缺失的小鼠在缺血-再灌注（I/R）损伤后发展出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依赖机制促进心肌细胞存活。我们提出以下三个具体目标来验证这一假设：具体目标1：确定细胞珠蛋白在维持心肌细胞稳态中的作用。具体目标2：明确细胞珠蛋白在适应不良心脏重塑中的作用。具体目标3：明确细胞球蛋白-p53相互作用对心肌细胞存活的意义。我们所提出的研究的成功完成将提供对调节心肌细胞存活的新型信号通路的深入了解，并可能为开发预防和/或逆转心力衰竭的新型治疗方法提供机会。因此，拟议的NIH R01研究资助申请与NIH和NHBLI的使命相关并保持一致。

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.