Converge of Pathways Regulating SMC Contractility

调节 SMC 收缩性途径的汇聚

• 批准号: 7312432
• 负责人: Avril V. Somlyo
• 金额: $ 56.66万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-18 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7312432
• 关键词: actins; cell cycle; cell migration; clinical research; coronary vessels; laboratory mouse; laboratory rabbit; mass spectrometry; microfilaments; muscle contraction; muscle disorders; muscle function; muscle relaxation; myocardium; myogenesis; myosin light chain kinase; myosins; phosphorylation; polymerase chain reaction; protein protein interaction; proteomics; small interfering RNA; tissue /cell culture; vascular smooth muscle; vertebrate embryology

The overall goal of this Project is to understand the complex processes that regulate contractility in vascular smooth muscle under physiological and pathophysiological conditions, which lead to high blood pressure, atherosclerosis, coronary restenosis, shock or cerebral vasospasm. Contractility is switched on in smooth muscle (SM) via phosphorylation of the regulatory light chain of myosin (RLC) and the level of phosphorylation is dependent upon the opposing activities of the Ca2+/calmodulin dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase, both of which can be regulated by upstream signaling pathways. We, with Project 2, have generated MLCK null mice, which are embryonic lethal, starting at E15.5 with some reaching term, but prior to this embryonic aortae or umbilical vessels display RLC phosphorylation and normal force development in response to Ca 2+. We will test the hypothesis that ubiquitously expressed SM MLCKs are critical for contraction, migration, filament and sarcomere formation in smooth and cardiac muscle respectively, as well as A404SMC "progenitor" cells (Project 2, Core A) and transformed proepicardial cells (Project 3, Core A); or alternatively that a compensatory kinase(s) accounts for the MLCK activity in MLCK null embryos or that another kinase normally predominants during embryonic development. Preliminary studies suggest that formation of the coronary vessels is defective in MLCK null embryos, thus, we will test the hypothesis that MLCKs are critical for migration of the epicardial cells of the proepicardial organ, the precursors of the coronary vessels with Project 3. The myosin motors underlying cell migration and contractility are also regulated and contribute to the SM contractile phenotype. These mechanisms will be explored using kinetic analysis with a novel recently synthesized fluorescent 3'-amino derivative of ATP. We will test the hypothesis that AM. ADP strongly bound crossbridges play a significant role in maintaining tonic force at low actomyosin activity, slow shortening velocity and low levels of RLC phosphorylation, characteristic features of SM myosins. The proposed studies require extensive interactions with the other projects and Core A and match the central theme of this PPG.

该项目的总体目标是了解在生理和病理生理条件下调节血管平滑肌收缩性的复杂过程，这些过程导致高血压、动脉粥样硬化、冠状动脉再狭窄、休克或脑血管痉挛。平滑肌 (SM) 中的收缩性通过肌球蛋白 (RLC) 调节轻链的磷酸化来开启，磷酸化水平取决于 Ca2+/钙调蛋白依赖性肌球蛋白轻链激酶 (MLCK) 和肌球蛋白轻链的相反活性磷酸酶，两者都可以受到上游信号通路的调节。我们通过项目 2 生成了 MLCK 无效小鼠，它们是胚胎致死的，从 E15.5 开始并达到一定足月，但在此之前，胚胎主动脉或脐血管表现出 RLC 磷酸化和响应 Ca 2+ 的正常力发育。我们将检验这样的假设：普遍表达的 SM MLCK 对平滑肌和心肌的收缩、迁移、丝状体和肌小节的形成以及 A404SMC“祖”细胞（项目 2，核心 A）和转化的心外膜细胞（项目 3）至关重要。 ，核心A）；或者，补偿性激酶负责 MLCK 无效胚胎中的 MLCK 活性，或者另一种激酶在胚胎发育过程中通常占主导地位。初步研究表明，MLCK 无效胚胎中冠状血管的形成存在缺陷，因此，我们将通过项目 3 检验 MLCK 对于心外膜器官（冠状血管的前身）的心外膜细胞的迁移至关重要的假设。细胞迁移和收缩性的运动也受到调节​​，并有助于 SM 收缩表型。将使用动力学分析和新颖的方法来探索这些机制 最近合成的 ATP 荧光 3'-氨基衍生物。我们将检验 AM 的假设。 ADP 强结合的横桥在低肌动球蛋白活性、缓慢的缩短速度和低水平的 RLC 磷酸化（SM 肌球蛋白的特征）下维持强直力方面发挥着重要作用。拟议的研究需要与其他项目和核心 A 进行广泛的互动，并符合本 PPG 的中心主题。