RSK Regulation of NFATc and Cardiac Myocyte Hypertrophy

描述（由申请人提供）：受压心脏的一个关键代偿反应是心肌细胞肥大。虽然心脏肥厚可以维持心输出量以应对壁应力升高，但持续的心脏肥厚往往伴随着不适应重构，最终导致心力衰竭。最近的证据表明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加深我们对RSK3在心脏重塑中的作用的理解，并有助于确定RSK3是否会成为一个有吸引力的治疗靶点，以衰减不适应的肥厚信号。特异性目的1：心肌细胞多分子信号复合物中RSK3结合位点的表征。我建议完善mAKAPp中主要RSK3结合位点的定位，并通过在哺乳动物异种细胞和细菌中的表达来测试RSK3是否直接与mAKAPp结合。特异性目的2：研究确定rsk3依赖性NFATc在肌细胞中的功能。RSK3可能转导肥厚性信号的潜在机制是通过促肥厚性NFATc（活化t细胞的核因子）转录因子家族的磷酸化。在体外诱导心肌细胞肥大的条件下，我将测试RSK3是否可以磷酸化并激活新生大鼠心肌细胞中的NFATc家族成员。rsk3依赖性磷酸化对NFATc功能的影响将通过荧光素酶报告基因测定来测量。此外，我将使用mAKAPp RSK3结合位点突变体来测试NFATc活性和心肌细胞肥大是否需要RSK3与mAKAPp信号体的关联。特异性目的3：体内心肌肥厚对RSK3的需求。为了测试心肌细胞肥大是否需要RSK3，我将描述先前产生的心脏特异性RSK3敲除小鼠的表型。我将采用左冠状动脉结扎诱导心肌梗死的方法挑战RSK3小鼠。心肌肥厚将在梗死后一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进行分析。此外，为了验证我的假设，即NFATc亚型的RSK3磷酸化对肥大很重要，将通过配对心脏特异性RSK来监测NFATc活性。nfat -荧光素酶指示剂小鼠。

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.