Prolyl hydroxylase oxygen sensor in Heart

描述(由申请人提供) 允许细胞感知和响应氧气的机制对于理解环境因素如何影响无数的生理、病理和发育过程是至关重要的。最近，细胞内O2-的感受/转导机制被确定为一类依赖O2的Pro羟基酶家族(PHD)。在低氧条件下，这些酶的羟基酶活性受到抑制。缺氧诱导因子-Lα不能羟化导致其蓄积。心血管系统对氧气表现出极高的敏感性，具有急性和慢性反应的特征。因此，候选人建议使用心脏细胞作为一个模型系统来探索这一氧气感知途径影响的具体机制。候选人的新数据表明，PHD氧气传感器可能在心脏的病理生理学中发挥重要作用。PHD的特异性抑制剂激活细胞的缺氧反应，并导致两种应激反应蛋白的诱导：一氧化氮合酶-2(NOS-2)和血红素加氧酶-1(HO-L)。这项建议的总体目标是开始探索PHD通路激活的后果及其在细胞对低氧应激反应中所起作用的性质。 具体目标1的目标是验证PHD抑制剂作为模型试剂的使用。新生儿心肌细胞培养将被用来检测慢性PHD途径的激活，以评估细胞对慢性缺氧的反应是否被PHD抑制剂概括。特殊目的2利用了一项发现，即一氧化氮合酶-2蛋白的高表达是由PHD途径激活诱导的。这一目标的目的是了解PHD抑制剂如何导致NOS-2的诱导。将通过启动子分析在转录水平上评估NOS-2的表达。这些研究还将探讨在信使核糖核酸水平上对一氧化氮合酶-2的可能调控。心脏对低氧最显著的急性反应是收缩能力下调。具体目标3的目的是批判性地检查PHD氧传感器在调节急性收缩功能障碍中所起的作用，这种急性收缩功能障碍被认为是缺血或缺氧性侮辱。

DESCRIPTION (provided by applicant) The mechanisms that allow cells to sense and respond to oxygen (O2) are of fundamental importance in understanding how environmental factors impact upon a myriad of physiological, pathological, and developmental processes. Recently the cellular O2-sensor/transducing mechanism has been identified as a family of O2-dependent prolyl hydroxylase enzymes (PHD). In hypoxic conditions the hydroxylase activity of these enzymes are inhibited. The failure to hydroxylate hypoxia-inducible factor-l alpha leads to its accumulation. The cardiovascular system displays exquisite sensitivity to O2 with a characteristic constellation of both acute and chronic responses. Thus, the candidate proposes using the heart cell as a model system to explore the specific mechanisms that are affected by this O2-sensing pathway. New data by the candidate suggests that the PHD oxygen sensor may play a large role in the pathophysiology of the heart. Specific inhibitors of PHD activate the cellular hypoxic response, and lead to induction of two stress-response proteins; nitric oxide synthase-2 (NOS-2) and heme oxygenase-1 (HO-l). The overall goal of this proposal is to begin to explore consequences of the PHD-pathway activation and the nature of its role in the cellular response to hypoxic stress. The goal of Specific Aim 1 is to validate the use of PHD inhibitors as model agents. Neonatal myocyte cultures will be used to examine chronic PHD-pathway activation in order to evaluate if cellular responses to chronic hypoxia are recapitulated by PHD inhibitors. Specific Aim 2 capitalizes on the finding that NOS-2 protein expression is highly induced by PHD-pathway activation. The goal of this aim is to understand how PHD inhibitors lead to NOS-2 induction. NOS-2 expression will be evaluated at the transcriptional level with promoter analysis. Possible regulation of NOS-2 at the mRNA level will also be addressed by these studies. The most prominent acute response of the heart to hypoxia is a down regulation of contractility. The goal of Specific Aim 3 is to critically examine the notion that the PHD oxygen sensor plays a role in mediating the acute contractile dysfunction that is seen with ischemic or hypoxic insult.