Mechanistic Study of Cleaved Serum Response Factor in Cardiac Hypertrophy

描述(由申请人提供)： 我最近的研究发现，血清反应因子(SRF)，一种强制性的生心转录因子，是人类衰竭心脏中一个重要的caspase-3靶点。SRF裂解导致显性负抑制物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，我培育了多个独立的转基因小鼠系，它们专门在心脏表达SRF-N。SRF-N高表达的小鼠发生扩张性肥厚性心肌病，最终死于心力衰竭。这提供了一种模拟人类心脏病进展的小鼠模型。基因芯片和定量聚合酶链式反应(Q-PCR)分析显示，在SRF-N转基因心脏中miR-133a和miR-1的表达显著下调，这与Mef2a、CaM和NFATc4的表达上调相一致。结合最近发现的作为miR-133a靶点的RhoA和CDC42，我们认为miR-133a和miR-1的失调以及随后一组肥大相关基因的上调可能是SRF-N介导的心肌病的潜在机制之一。这些初步结果支持了应用程序的中心假设，即显性负性SRF-N通过下调miR-133a和miR-1基因，引导心肌病的发生，并促进发展为明显的心力衰竭。提出了三个目标。目的研究转基因小鼠的表型特征。转基因小鼠将通过包括功能、形态和分子评估在内的全面研究进行评估。目的II和III阐明SRF-N介导心肌病的分子机制。将评估这两个心脏microRNAs的失调。将对一组miR-133a和miR-1调控的靶基因进行评估。将验证针对miR-133a表达的两个新增强子和针对miR-133a的一个新靶点。将进行抢救性实验，以测试引入miR-133a和-1是否可以纠正/挽救小鼠的心脏表型。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介导的完整心脏心肌病；2)鉴定了两个调节miR-133a表达的SRF依赖的增强子；以及3)阐明了指导疾病进展的分子机制。 公共卫生相关性： 本研究重点阐述了显性负性心源性转录因子SRF-N(血清反应因子N末端)在心力衰竭中的作用机制。

DESCRIPTION (provided by applicant): My recent study 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 inhibitor, 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, I generated multiple independent lines of transgenic mice that expressed SRF-N specifically in the heart. Mice with high expression level of SRF-N developed a dilated, hypertrophic cardiomyopathy and eventually died of heart failure. This provides a mouse model that mimics the progression of human heart disease. Microarray and quantitative PCR (Q-PCR) analyses revealed a significant downregulation of miR-133a and miR-1 in the SRF-N transgenic hearts, which coincided with overt upregulation of Mef2a, CaM and NFATc4. Together with recent discovery of RhoA and Cdc42 as miR-133a targets, we believe that the dysregulation of miR-133a and miR-1 and the consequent upregulation of a group of hypertrophy-associated genes may constitute one of the potential mechanisms contributing to SRF-N-mediated cardiomyopathy. These preliminary results underpin the application's central hypothesis that the dominant negative SRF-N directs the onset of cardiomyopathy and facilitates the progression to overt heart failure through the downregulation of miR-133a and miR-1 gene. Three aims are proposed. Aim I is to characterize the transgenic mouse phenotype. The transgenic mice will be assessed by comprehensive studies including functional, morphological and molecular assessments. Aim II and III are to elucidate the molecular mechanism of SRF-N-mediated cardiomyopathy. Dysregulation of the two cardiac microRNAs will be assessed. A group of miR-133a and miR-1 regulated target genes will be evaluated. Two new enhancers directed for miR-133a expression and one new target for miR-133a will be verified. Rescue experiments will be conducted to test if the introduction of miR-133a and -1 could correct/rescue the murine heart phenotype. 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 cardiomyopathy in intact heart; 2) the identification of two SRF-dependent enhancers regulating miR-133a expression; and 3) the elucidation of the molecular mechanism directing disease progression. PUBLIC HEALTH RELEVANCE: The study is focused on the mechanistic view of dominant negative cardiogenic transcription factor SRF-N (N-terminus of serum response factor) in propelling heart failure.