RSK Regulation of NFATc and Cardiac Myocyte Hypertrophy

描述(申请人提供)：应激性心脏的一个关键的代偿反应是心肌细胞肥大。尽管心肌肥厚可以在室壁应力升高时维持心输出量，但持续性心肌肥厚往往伴随着适应性不良的重塑，最终可导致心力衰竭。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是否会成为减弱不良适应肥厚信号的有吸引力的治疗靶点。具体目标1：心肌细胞多分子信号复合体中RSK3结合位点的特征。我建议完善mAKAPp中主要的RSK3结合位点的定位，并通过在哺乳动物异源细胞和细菌中的表达来测试RSK3是否直接与mAKAPp结合。特定目的2：研究心肌细胞中依赖RSK3的NFATc功能。RSK3可能转导肥大信号的一个潜在机制是通过促进肥大的转录因子家族NFATc的磷酸化。我将测试在体外诱导心肌细胞肥大的条件下，RSK3是否能够磷酸化和激活新生大鼠心肌细胞中的NFATc家族成员。依赖RSK3的磷酸化对NFATc功能的影响将通过荧光素酶报告分析来衡量。此外，我将使用mAKAPp RSK3结合位点突变体来测试RSK3与mAKAPp信号小体的关联是否是NFATc活性和心肌细胞肥大所必需的。具体目的3：在体心肌肥大对RSK3的需求。为了测试RSK3是否是心肌细胞肥大所必需的，我将对先前产生的心脏特异性RSK3基因敲除小鼠的表型进行表征。我将通过结扎左冠状动脉和诱导心肌梗死来挑战RSK3小鼠。心肌梗死后1个月行超声心动图、血流动力学分析、大体及组织病理学检查。此外，为了解决我的假设，即NFATc亚型的RSK3磷酸化对肥厚很重要，将通过与心脏特异的RSK交配来在体内监测NFATc的活性。小鼠给NFAT-荧光素酶指示剂小鼠。 心脏肥厚是心力衰竭的主要风险因素，心力衰竭是一种对公众健康具有重大意义的综合征，影响着520万美国人。更好地了解控制心肌细胞肥大的机制可能会使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.