Role of p67phox in myocardial hypertrophy

说明(申请人提供)：NADPH氧化酶(NOX)似乎在心肌肥厚的发展和随后的心力衰竭进展中发挥作用。然而，NOX在心肌细胞中的信号转导机制尚不清楚。本研究的长期目标是了解NOX在过度肾上腺素能刺激和血流动力学压力超负荷所致心肌肥厚和心力衰竭的发生发展中的信号机制。我们的初步数据表明，NOx的主要胞浆亚基p67Phox通过一种新的不依赖于活性氧(ROS)的机制介导心肌肥厚的发展。基于这些数据，我们假设NOx/p67Phox介导了包括11-AR刺激和血流动力学压力超载在内的肥厚刺激诱导的心肌肥厚，其信号转导功能涉及一种新的ROS非依赖性机制。为了验证这一假设，我们提出了以下三个特定目标：在特定目标1中，我们将确定NOx/p67Phox是否在体外介导11-肾上腺素能受体(11-AR)刺激的心肌肥厚，以及这是否涉及一种新的ROS非依赖性信号机制。NOx胞质亚基p67Phox和p47Phox的显性负性突变体和野生型结构。突变或WT结构将在培养的成年大鼠心室肌细胞(ARVM)中过表达。这些突变体或WT构建体对11-AR刺激诱导的肥大和NOX酶活性的影响将被确定。在这一目标中，还将确定p67Phox和MEK-ERK级联之间潜在的蛋白质-蛋白质相互作用。在特定的目标2中，我们将在体内测试p67Phox是否介导了11-AR刺激的心肌肥大。我们将使用新产生的具有心脏特异过表达的dN p67Phox突变体的杂合的dN-p67转基因(TG)小鼠来产生纯合的dN-p67转基因小鼠。由于心脏特异表达11B-AR突变体(CAM-11B-AR)的转基因小鼠心肌肥厚，我们将这两个TG系(dN-p67和cAM-11B-AR)进行杂交，以研究dN p67Phox突变体蛋白在心脏过度表达对11B-AR诱导的在体心肌肥大的影响。在特定的目标3中，我们将在体内测试心肌p67Phox在介导血流动力学超负荷诱导的心肌肥厚中的作用。纯合子的DN-p67TG小鼠和年龄和性别匹配的WT对照组小鼠将受到升主动脉收缩(AAC)引起的慢性压力超负荷。我们将研究AAC或假手术的TG和WT小鼠的心脏表型和功能，以确定p67Phox在体内介导慢性压力超负荷诱导的心肌肥厚中的作用。我们的研究将对NOx/p67Phox在心肌肥厚发展中的作用和新机制提供新的理解，从而为治疗充血性心力衰竭提供新的治疗方法。公共卫生相关性我们的研究将为我们提供新的认识，了解p67Phox和NADPH氧化酶在心脏肥大发展过程中的新的病理机制，而肥大可能导致主要的心血管疾病，心力衰竭。因此，我们的研究将与心力衰竭的一种常见原因直接相关，这种原因具有很高的死亡率和致残率，是对公众健康的主要威胁。

DESCRIPTION (provided by applicant): NADPH oxidase (NOX) appears to play a role in the development of myocardial hypertrophy and subsequent progression of heart failure. However, the signaling mechanisms of NOX in cardiac myocytes is unknown. The long-term goal of this research is to understand the signaling mechnisms of NOX in the development of myocardial hypertrophy and heart failure induced by excessive adrenergic stimulation and hemodynamic pressure overload. Our prelminary data indicate that p67phox, the major cytosolic subunit of NOX, mediates the development of myocardial hypertrophy through a novel reactive oxygen species (ROS) - independent mechanism. Based on these data, we hypothesize that NOX/p67phox mediates myocardial hypertrophy that is induced by hypertrophic stimuli including 11-AR-stimulation and hemodynamic pressure overload, and its signaling function involve a novel ROS-independent mechanism. To test this hypothesis, we proposed the following three Specific Aims: In Specific Aim 1, we will determine whether NOX/p67phox mediates 11-adrenergic receptor (11-AR)- stimulated myocardial hypertrophy and whether this involves a novel ROS-independent signaling mechanism in vitro. Dominant negative (DN) mutants and wild type (WT) contstructs of two NOX cytosolic subunits, p67phox and p47phox. Mutants or WT constructs will be overexpressed in cultured adult rat ventricular myocytes (ARVM). The effects of these mutants or WT constructs on 11-AR stimulation-induced hypertrophy and NOX enzyme activity will be determined. The potential protein-protein interaction between p67phox and MEK-ERK cascade will also be determined in this aim. In Specific Aim 2, we will test if p67phox mediates 11-AR-stimulated cardiac hypertrophy in vivo. We will use the newly generated heterozygous DN-p67 transgenic (TG) mice with cardiac-specific overexpression of the DN p67phox mutant to generate homozygous DN-p67 TG mice. Since the commercially available TG mice with cardiac-specific overexpression of a constitutively activated mutant of 11B-AR (CAM-11B-AR) develop cardiac hypertrophy, we will crossbreed these two TG lines (DN-p67 and CAM-11B-AR) to study the effect of overexperssion of DN p67phox mutant protein in the heart on 11B-AR-induced myocardial hypertrophy in vivo. In Specific Aim 3, we will test the role of myocardial p67phox in mediating hemodynamic overload- induced myocardial hypertrophy in vivo. The homozygous DN-p67 TG mice and age- and gender-matched WT control mice will be subjected to chronic pressure overload caused by ascending aorta constriction (AAC). The cardiac phenotypes and function of these TG and WT mice with AAC or sham operation will be studied to determine the role of p67phox in mediating chronic pressure overload-induced myocardial hypertrophy in vivo. Our studies will provide new understanding of the role and novel mechanisms of NOX/p67phox in the development of cardiac hypertrophy that should lead to novel treatments for congestive heart failure. PUBLIC HEALTH RELEVANCE Our studies will provide new understanding of the novel pathological mechanisms of p67phox and NADPH oxidase in the development of hypertrophy in the heart, which could lead to the major cardiovascular disease, heart failure. Therefore, our research will have direct relevance to a common cause of heart failure, which has a high mortality and disability rate and is a major threat to the public health.