Mechanism of SRF-N-mediated Cardiac Suppression

描述（由申请人提供）：我的实验室发现血清反应因子（SRF），一种强制性的心源性转录因子，是人类衰竭心脏中重要的caspase-3靶点。SRF的裂解导致显性负转录因子SRF- n （SRF的n端）的产生。这一新发现具有挑衅性，并提出了SRF-N在心功能障碍中的潜在致病作用的问题。为了解决这个问题，我们产生了多个独立的转基因小鼠系，在心脏中表达SRF-N。高表达SRF-N的小鼠表现出与人类衰竭心肌相当的水平，并发展为两期心肌病表型：适应性肥厚，随后是扩张型心肌病心衰。这提供了一种模拟人类心脏病进展的新型小鼠模型。微阵列和定量PCR （Q-PCR）分析显示，在转基因心脏中miR-133a显著下调，这与两个可能的miR-133a靶基因NFATc4（活化T细胞的核因子4）和CamK22 （Ca2????）的强烈上调相吻合依赖性蛋白激酶2)可能决定心脏表型。这些研究支持了该应用程序的中心假设，即显性负SRF-N通过抑制miR-133a基因上调肥厚基因，指导心脏肥厚的发生，并促进公开性心力衰竭的进展。提出了两个主要目标。目的1是确定SRF-N在完整心脏中的致病作用。突变小鼠将在基础和血流动力学过载条件下进行严格评估。分析心脏功能、形态学和组织学的变化以及心脏重塑基因在肥大的起始、发展和失代偿三个阶段的表达。通过比较低、中、高表达SRF-N的小鼠，进一步确定SRF-N的负面影响。我们有初步数据表明，通过SRF-N抑制miR-133a上调促肥厚性NFATc4和CamK22基因可能与SRF-N介导的心肌病有关。因此，Aim II的重点是1)验证SRF-N-> miR-133a-> NFATc4和CamK22->肥厚信号通路；2)阻断部分通路，观察小鼠表型是否得到纠正。该应用的最终结果将是在主导的负SRF-N和心力衰竭的发展之间建立直接联系。其新颖性包括1)在完整心脏中证明srf - n介导的肥厚性心肌病；2)鉴定两个srf依赖性增强子调控miR-133a表达；3)阐明两个新的miR-133a靶基因指导疾病进展。

DESCRIPTION (provided by applicant): My laboratory identified serum response factor (SRF), an obligatory cardiogenic transcription factor, as a prominent caspase-3 target in human failing hearts. SRF cleavage led to the generation of a dominant negative transcription factor, SRF-N (N-terminus of SRF). This novel discovery is provocative, and raises the question of potential pathogenic role of SRF-N in cardiac dysfunction. To address this question, we generated multiple independent lines of transgenic mice expressing SRF-N in the heart. Mice with high expression SRF-N showed levels comparable to those in human failing myocardium, and developed a two-stage cardiomyopathy phenotype: adaptive hypertrophy followed by heart failure with dilated cardiomyopathy. This provides a novel mouse model that mimics the progression of human heart disease. Microarray and quantitative PCR (Q-PCR) analyses revealed a significant down regulation of miR-133a in the transgenic hearts, which coincided with a robust up regulation of two likely miR-133a target genes NFATc4 (nuclear factor of activated T cells 4) and CamK22 (Ca2????dependent protein kinase 2) that may determine the cardiac phenotype. These studies underpin the application's central hypothesis that the dominant negative SRF-N directs the onset of cardiac hypertrophy and facilitates the progression to overt heart failure through the up regulation of hypertrophic genes by repressing miR-133a gene. Two main aims are proposed. The Aim I is to determine the pathogenic impact of SRF-N in intact heart. The mutant mice will be critically evaluated under basal and hemodynamic overload conditions. Analysis in cardiac function, changes in morphology and histology and expression of cardiac remodeling genes at three stages: initiation, development and decompensation of hypertrophy will be assessed. The negative impact of SRF-N will be further determined by comparing among the mice expressing low, intermediate and high levels of SRF-N. We have preliminary data suggesting that the up regulation of pro-hypertrophic NFATc4 and CamK22 genes by repressing miR-133a through SRF-N may be associated with SRF-N-mediated cardiomyopathy. The Aim II, therefore, is focused on 1) the verification of this SRF-N-> miR-133a-> NFATc4 and CamK22-> hypertrophy signaling pathway; 2) by blocking parts of this pathway to see if the mouse phenotype is corrected. The ultimate outcome of the application will be to establish a direct link between the dominant negative SRF-N and the development of heart failure. The novelty includes 1) the demonstration of SRF-N-mediated hypertrophic cardiomyopathy in intact heart; 2) the identification of two SRF-dependent enhancers regulating miR-133a expression; and 3) the elucidation of two new miR-133a target genes directing disease progression. PUBLIC HEALTH RELEVANCE: Project narrative: The study is to determine the negative role of the transcription factor SRF-N in intact heart, and to verify the preliminarily identified molecular signaling pathway leading to the SRF-N-mediated cardiac suppression. The genetic-manipulated murine mice are applied.