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)，一种必需的生心转录因子，是人类衰竭心脏中一个重要的caspase-3靶点。SRF裂解导致显性负转录因子SRF-N(SRF的N-末端)的产生。这一新的发现具有挑衅性，并提出了SRF-N在心脏功能障碍中的潜在致病作用的问题。为了解决这个问题，我们培育了多个在心脏中表达SRF-N的转基因小鼠。高表达SRF-N的小鼠的水平与人类衰竭心肌中的水平相当，并发展出两个阶段的心肌病表型：适应性肥厚，随后心力衰竭伴扩张型心肌病。这提供了一种新的小鼠模型，可以模拟人类心脏病的进展。基因芯片和定量聚合酶链式反应(Q-PCR)分析显示，在转基因心脏中miR-133a的表达显著下调，与两个可能决定心脏表型的可能的miR-133a靶基因NFATc4(活化T细胞核因子4)和CamK22(钙依赖蛋白激酶2)的强劲上调相一致。这些研究支持了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，引导心肌肥厚的开始，并促进进展为显性心力衰竭。提出了两个主要目标。目的I是确定SRF-N对完整心脏的致病影响。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。本课程将评估心脏肥厚发生、发展和失代偿三个阶段的心功能分析、形态和组织学变化以及心脏重塑基因的表达。通过比较表达低水平、中等水平和高水平SRF-N的小鼠，将进一步确定SRF-N的负面影响。我们有初步的数据表明，通过SRF-N抑制miR-133a，促进肥厚性NFATc4和CamK22基因的上调可能与SRF-N介导的心肌病有关。因此，目标II集中在1)验证SRF-N-&gt；miR-133a-&gt；NFATc4和CamK22-&gt；肥大信号通路；2)通过阻断这一通路的部分片段，看看小鼠的表型是否得到纠正。该应用的最终结果将是在显性负性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)证实了SRF-N介导的完整心脏肥厚性心肌病；2)鉴定了两个调节miR-133a表达的依赖SRF的增强子；以及3)阐明了两个新的miR-133a靶基因，指导疾病的进展。

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.