RSK Regulation of NFATc and Cardiac Myocyte Hypertrophy

描述（由申请人提供）：应激心脏的一个关键代偿反应是肌细胞肥大。虽然心脏肥大可以维持心输出量，以应对升高的室壁应力，但持续的心脏肥大往往伴随着适应不良的重塑，最终可能导致心力衰竭。最近的研究表明，p90核糖体S6激酶（RSK）可能是心肌细胞肥大的重要调节因子。我提出的研究将进一步了解RSK 3在心脏重塑中的作用，并帮助确定RSK 3是否会成为一个有吸引力的治疗靶点，用于减弱适应不良的肥大信号。具体目标1：心肌细胞多分子信号复合物内RSK 3结合位点的表征。我建议完善mAKAPp中主要RSK 3结合位点的映射，并使用哺乳动物异源细胞和细菌中的表达来测试RSK 3是否直接与mAKAPp结合。具体目标2：确定肌细胞中RSK 3依赖性NFATc功能的研究。RSK 3可能抑制肥大信号传导的潜在机制是通过促肥大NFATc（活化T细胞的核因子）转录因子家族的磷酸化。我将测试RSK 3是否可以磷酸化和激活NFATc家族成员在新生大鼠心肌细胞的条件下，引起心肌细胞肥大在体外。RSK 3依赖性磷酸化对NFATc功能的影响将使用荧光素酶报告基因测定来测量。此外，我将使用mAKAPp RSK 3结合位点突变体来测试RSK 3与mAKAPp信号体的结合是否是NFATc活性和肌细胞肥大所必需的。具体目标3：体内心肌肥大对RSK 3的需求。为了测试RSK 3是否是心肌细胞肥大所必需的，我将表征先前产生的心脏特异性RSK 3敲除小鼠的表型。我将通过左冠状动脉结扎和诱导心肌梗死来激发RSK 3小鼠。将在梗死后1个月通过超声心动图和血流动力学分析以及大体和组织病理学分析心脏肥大。此外，为了解决我的假设，RSK 3磷酸化的NFATc亚型是重要的肥大，NFATc的活动将在体内监测交配的心脏特异性RSK？小鼠与NFAT-荧光素酶指示小鼠的比较。 心脏肥大是心力衰竭的主要危险因素，心力衰竭是一种影响520万美国人的重大公共健康意义综合征。更好地了解控制心肌细胞肥大的机制可能会允许更好的治疗方案，降低死亡率。

DESCRIPTION (provided by applicant): A key compensatory response in the stressed heart is myocyte hypertrophy. Although cardiac hypertrophy can maintain cardiac output in response to elevated wall stress, sustained cardiac hypertrophy is often accompanied by maladaptive remodeling which can ultimately lead to heart failure. Recent evidence suggests that p90 ribosomal S6 kinase (RSK) might be an important regulator of cardiac myocyte hypertrophy. My proposed studies will further our understanding of the role of RSK3 in cardiac remodeling and help determine whether RSK3 would make an attractive therapeutic target for the attenuation of maladaptive hypertrophic signaling. Specific Aim 1: Characterization of a RSK3 binding site within a cardiac myocyte multimolecular signaling complex. I propose to refine the mapping of the predominant RSK3 binding site in mAKAPp and to test whether RSK3 binds to mAKAPp directly using expression in mammalian heterologous cells and bacteria. Specific Aim 2: Studies determining RSK3-dependent NFATc function in myocytes. A potential mechanism by which RSK3 may transduce hypertrophic signaling is through the phosphorylation of the pro-hypertrophic NFATc (nuclear factor of activated T-cells) transcription factor family. I will test whether RSK3 can phosphorylate and activate NFATc family members in neonatal rat cardiac myocytes under conditions that elicit cardiac myocyte hypertrophy in vitro. The effect of RSK3-dependent phosphorylation on NFATc function will be measured using luciferase reporter assays. In addition, I will use mAKAPp RSK3-binding site mutants to test whether RSK3 association with the mAKAPp signalosome is required for NFATc activity and myocyte hypertrophy. Specific Aim 3: The requirement for RSK3 in cardiac hypertrophy in vivo. In order to test whether RSK3 is required for myocyte hypertrophy, I will characterize the phenotype of cardiac-specific RSK3 knock-out mice that have been previously generated. I will challenge the RSK3 mice by left coronary artery ligation and induction of myocardial infarction. Cardiac hypertrophy will be analyzed at one month post-infarction by echocardiography and hemodynamic analysis and by gross and histopathology. Further, in order to address my hypothesis that RSK3 phosphorylation of NFATc isoforms is important for hypertrophy, NFATc activity will be monitored in vivo by mating the cardiac-specific RSK?'' mice to NFAT-luciferase indicator mice. Cardiac hypertrophy is a leading risk factor for heart failure, and heart failure is a syndrome of major public heath significance affecting 5.2 million US individuals. A better understanding of the mechanisms controlling myocyte hypertrophy may allow for better therapeutic regimens with decreased mortality.