Cytoglobin: A stress-responsive hemoprotein modulating cardiomyocyte survival

描述(申请人提供)：心力衰竭是美国心血管疾病死亡的主要原因，通常是由于高血压或心肌梗死(MI)导致的心脏重构不良所致。尽管在过去的十年里，我们在治疗心力衰竭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，识别阻止或逆转不良适应性心脏重构过程的候选蛋白质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珠蛋白(Cygb)是一种应激反应的血红蛋白，在各种应激条件下(如缺氧、缺血和压力超负荷)，其水平在成人心脏中升高。在培养的心肌细胞中，Cygb的过表达保护它们免受氧化应激，而Cygb的敲除会导致细胞凋亡增加。最初，我们假设Cygb通过所有血红蛋白固有的抗氧化机制来保护心肌细胞；然而，现在我们有广泛的证据表明，Cygb还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作用关系。染色质免疫沉淀实验证明，P53和Cygb共同作用于经典的P53靶启动子。利用重组蛋白质，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，在体外重组转录实验中，重组Cygb抑制P53的转录活性。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基因缺失会导致扩张型心肌病。同样，与受伤的对照组小鼠相比，出生时心脏特异缺失Cygb的小鼠在缺血再灌注(I/R)损伤后发展为更明显的扩张性心肌病。我们的中心假设是Cygb通过抗氧化和P53依赖的机制促进心肌细胞存活。我们提出了以下三个特定目标来验证这一假说：特定目标1：确定细胞球蛋白在维持心肌细胞内稳中的作用。具体目标2：明确细胞球蛋白在不良适应性心脏重塑中的作用。具体目标3：明确细胞珠蛋白-P53相互作用对心肌细胞存活的意义。我们建议的研究的成功完成将为调节心肌细胞存活的新的信号通路提供洞察力，并可能为开发预防和/或逆转心力衰竭的新的治疗方法提供机会。因此，拟议的NIH R01研究补助金申请与NIH和NHBLI的任务相关并保持一致。

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.