Fibrogenic Role of ROCK delta1 and Mechanism in Cardiac Remodeling

ROCK delta1 的纤维化作用及其在心脏重构中的机制

• 批准号: 7865741
• 负责人: Jiang Chang
• 金额: $ 36.63万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7865741
• 关键词: Actins; Address; Adenoviruses; Angiotensin II; Animals; Attenuated; Binding; Bioinformatics; Biological Assay; Cardiac; Cardiac Myocytes; Cardiomyopathies; Caspase; Cell Culture Techniques; Collagen; Culture Media; Data; Depressed mood; Doppler Echocardiography; EMSA; Elements; Endothelin-1; Enhancers; Enzyme-Linked Immunosorbent Assay; Fibrosis; Figs - dietary; Gel; Genetic; Goals; Heart; Heart Diseases; Histology; Human; Indium; Interleukin-6; Knockout Mice; LacZ Genes; Link; Luciferases; Measurement; Mediating; Molecular; Morphology; Mus; Mutant Strains Mice; Nuclear; Outcome; Phenotype; Phosphorylation; Platelet Factor 4; Proteins; Regulation; Regulatory Element; Rho-associated kinase; Role; Serum; Serum Response Factor; Signal Pathway; Signal Transduction; Smad Proteins; Smooth Muscle Actin Staining Method; Stimulus; Stress; Testing; Tissues; Transforming Growth Factors; Transgenic Mice; Transgenic Organisms; Up-Regulation; caspase-3; chromatin immunoprecipitation; connective tissue growth factor; constriction; cytokine; experimental analysis; fasudil; in vitro testing; in vivo; inhibitor/antagonist; innovation; kinase inhibitor; mutant; novel; osteopontin; pressure; promoter; public health relevance; research study; response; rho; therapeutic target; transcription factor

DESCRIPTION (provided by applicant): We identified Rho kinase ROCK1 as caspase-3 target in human failing hearts. The cleavage resulted in a constitutively active Rho kinase, ROCK?1. We also demonstrated that genetic deletion of ROCK1 inhibited stress-induced cardiac fibrosis. However, the function of ROCK?1 and molecular signaling linking ROCK1 to cardiac fibrosis remain obscure. The goal of the study is to determine the fibrogenic role of ROCK?1 and investigate molecular mechanism of ROCK?1-mediated cardiac fibrosis. To address these questions, we generated transgenic mice expressing ROCK?1 in heart to mimic human heart disease. Overt cardiac fibrosis was observed with marked upregulation of TGF¿1 in the transgenic mice. Since activation of Rho kinase increased SRF activity, we assessed the transgenic heart and found manifest increase in SRF activity. Our preliminary results suggested SRF as a potential regulator of TGF¿1. We also found robust increases in NF-?B expression and activity in the mice. Therefore, the central hypothesis is that constitutive activation of ROCK1 in cardiomyocytes is sufficient to result in cardiac fibrosis by upregulating TGF¿ signaling and other pro-fibrotic cytokines through activation of SRF and NF-?B, respectively. Three aims will be completed. Aim I is to determine the pro-fibrotic effect of ROCK?1 in intact heart. The transgenic mice will be studied under basal and stress challenging conditions. The fibrogenic phenotype will be determined in two mouse lines with high and low expression level of ROCK?1. A rescue experiment by Rho kinase inhibitor will be conducted. Aim II is to elucidate the molecular mechanisms of ROCK?1-mediated cardiac fibrosis. The signaling pathway linked between Rho kinase and fibrotic response has been proposed with a considerable amount of preliminary data. The proposed mechanism includes the upregulation of TGF¿1 and NF-?B-mediated cytokines. The hypothesis will be tested in vitro in cardiomyocytes and in vivo in the transgenic mice. To investigate SRF-directed TGF¿1 regulation, the identified cis elements in TGF¿1 promoter/enhancer region will be verified by extensive experiments including 1) luciferase, EMSA and CHIP assay; 2) through transgenic mice expressing lacZ driven by either the wild or mutant cis elements; 3) by analyzing expression of TGF¿1 in SRF null mouse heart, where the decrease in TGF?1 level is expected. Aim III is to determine if the loss of endogenous ROCK1 inhibitor, Rnd3, recapitulates ROCK?1-mediated fibrotic cardiomyopathy. The fibrotic phenotype, Rho kinase activity, TGF? and NF-?B signaling will be assessed under normal and stress challenging conditions. The outcome of the proposal will be to establish links between the activation of Rho kinase, TGF¿1 and NF-?B signaling in fibrotic cardiac remodeling. The innovation of the proposal includes 1) demonstration of fibrogenic role of ROCK?1; 2) elucidation of the ROCK1->SRF->TGF?1->fibrosis and the ROCK1->NF-?B->cytokines->fibrosis signaling pathways; 3) revelation the role of Rnd3 in cardiac remodeling; and 4) implication of manipulating Rho kinase activity and caspase cleavage as candidate therapeutic targets. PUBLIC HEALTH RELEVANCE: The study is to determine the fibrogenic role of the constitutively active ROCK?1 in intact heart, and to elucidate the molecular mechanism involved in Rho kinase-mediated cardiac fibrosis. The genetic-manipulated murine mice are applied.

描述(申请人提供)：我们在人类衰竭的心脏中鉴定了Rho激酶ROCK1为caspase-3靶标。裂解导致了Rho-1的结构性活性，ROCK？1。我们还证明了ROCK1的基因缺失抑制了应激诱导的心肌纤维化。然而，ROCK？1的功能和将ROCK1与心肌纤维化联系起来的分子信号仍不清楚。本研究的目的是确定ROCK？1的促纤维化作用，并探讨ROCK？1介导的心肌纤维化的分子机制。为了解决这些问题，我们培育了在心脏中表达ROCK-1的转基因小鼠，以模拟人类心脏病。在转基因小鼠中观察到明显的心肌纤维化，并显著上调了转化生长因子β1。由于Rho激酶的激活增加了SRF的活性，我们对转基因心脏进行了评估，发现SRF的活性明显增加。我们的初步结果表明，SRF是一种潜在的转化生长因子1的调节因子。我们还发现，在小鼠中，核因子-βB的表达和活性显著增加。因此，中心假说是，心肌细胞中ROCK1的结构性激活足以通过分别通过激活SRF和NF-β上调转化生长因子和其他促纤维化细胞因子而导致心肌纤维化。三个目标将会实现。目的研究ROCK-1对正常心脏的促纤维化作用。转基因小鼠将在基础和应激挑战条件下进行研究。将在ROCK？1高表达和低表达的两个小鼠系中确定纤维化表型。将进行Rho激酶抑制剂的救治实验。目的II阐明ROCK-1介导的心肌纤维化的分子机制。Rho激酶与纤维化反应之间的信号通路已经被提出，并有大量的初步数据。其机制包括上调转化生长因子β1和核因子βB介导的细胞因子。这一假说将在体外心肌细胞和转基因小鼠体内进行验证。为了研究SRF对转化生长因子β1的调控，将通过广泛的实验来验证已确定的转化生长因子1启动子/增强子区域的顺式元件，包括1)荧光素酶、EMSA和芯片检测；2)通过野生型和突变型顺式元件驱动的表达LacZ的转基因小鼠；3)通过分析转化生长因子β1在SRF缺失小鼠心脏中的表达，预计转化生长因子β1的水平将降低。目的III确定内源性ROCK1抑制剂RND3的缺失是否重演ROCK-1介导的纤维性心肌病。肝纤维化表型、Rho激酶活性、转化生长因子？并将在正常和应激挑战条件下评估核因子-βB信号。该提案的结果将是在纤维化心脏重塑中Rho激酶、转化生长因子β1的激活和核因子-β信号之间建立联系。该提案的创新包括：1)ROCK？1-GT；2)阐明ROCK-1-GT；SRF-&GT；TGF？1-&GT；纤维化及ROCK1--GT；NF-B-&GT；细胞因子-GT；纤维化信号通路；3)揭示RND3在心脏重塑中的作用；4)调控Rho激酶活性和caspase裂解作为候选治疗靶点的意义。 公共卫生相关性：本研究旨在确定活性成分ROCK？1在完整心脏中的致纤维化作用，并阐明Rho激酶介导的心肌纤维化的分子机制。应用了基因操纵的小鼠。