Mechanism of SRF-N-mediated Cardiac Suppression

描述（由申请人提供）： 我的实验室鉴定了血清反应因子（SRF），一种强制性心源性转录因子，作为人类衰竭心脏中的一个突出的caspase-3靶标。SRF切割导致显性负性转录因子SRF-N（SRF的N末端）的产生。这一新的发现是挑衅性的，并提出了SRF-N在心功能不全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系的转基因小鼠在心脏中表达SRF-N。具有高表达SRF-N的小鼠显示出与人类衰竭心肌中的水平相当的水平，并发展出两阶段心肌病表型：适应性肥大，随后是心力衰竭伴扩张型心肌病。这提供了一种新的小鼠模型，模拟人类心脏病的进展。微阵列和定量PCR（Q-PCR）分析显示，在转基因心脏中，miR-133 a显著下调，这与miR-133 a的两个可能的靶基因NFATc 4（活化T细胞核因子4）和CamK 22（Ca 2+）的显著上调相一致。依赖性蛋白激酶2），可能决定心脏表型。这些研究支持了本申请的中心假设，即显性负性SRF-N通过抑制miR-133 a基因上调肥大基因，指导心脏肥大的发生，并促进向明显心力衰竭的进展。 提出了两个主要目标。目的I是确定SRF-N在完整心脏中的致病影响。将在基础和血流动力学超负荷条件下对突变小鼠进行严格评价。将评估心脏功能分析、形态学和组织学变化以及三个阶段（肥大的开始、发展和失代偿）的心脏重塑基因表达。通过比较表达低、中和高水平SRF-N的小鼠，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-133 a上调促肥大NFATc 4和CamK 22基因可能与SRF-N介导的心肌病有关。因此，目标II的重点是1）验证这种SRF-N-> miR-133 a-> NFATc 4和CamK 22->肥大信号通路; 2）通过阻断该通路的一部分来观察小鼠表型是否得到纠正。 该应用的最终结果将是建立显性阴性SRF-N与心力衰竭发展之间的直接联系。新奇包括：1）在完整心脏中证实SRF-N介导的肥厚型心肌病; 2）鉴定两种调节miR-133 a表达的SRF依赖性增强子; 3）阐明两种指导疾病进展的新miR-133 a靶基因。

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.