Converge of Pathways Regulating SMC Contractility

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能力的复杂过程，这些过程会导致高血压、动脉粥样硬化、冠状动脉再狭窄、休克或脑血管痉挛。肌球蛋白轻链(RLC)的磷酸化水平依赖于钙/钙调蛋白依赖的肌球蛋白轻链激酶(MLCK)和肌球蛋白轻链磷酸酶(MLCK)的相对活性，两者均可通过上游信号通路进行调节。与项目2一起，我们已经产生了MLCK缺失的小鼠，它们是胚胎致命的，从E15.5开始，一些达到了期末，但在此之前，胚胎主动脉或脐带血管显示RLC磷酸化和正常的力量发展对钙的反应。我们将检验一种假设，即普遍表达的SM MLCK分别对平滑肌和心肌以及A404SMC的“祖细胞”细胞(项目2，核心A)和转化的心外膜细胞(项目3，核心A)的收缩、迁移、细丝和肌节形成起关键作用；或者，另一种假设是，在MLCK缺失的胚胎中，MLCK活性是由一种补偿激酶(S)解释的，或者在胚胎发育过程中，另一种激酶通常占主导地位。初步研究表明，在MLCK缺失的胚胎中，冠状血管的形成是有缺陷的，因此，我们将检验这一假设，即MLCKs对于项目3中冠脉的前体--心外膜器官的心外膜细胞的迁移是至关重要的。支持细胞迁移和收缩的肌球蛋白马达也受到调节，并对SM收缩表型做出贡献。这些机制将用一种新的动力学分析来探索 最近合成了3‘-氨基荧光的三磷酸腺苷衍生物。我们将测试AM的假设ADP强结合的交叉桥在肌动球蛋白活性低、缩短速度慢和RLC磷酸化水平低的情况下对维持紧张力起着重要作用，这是SM肌球蛋白的特征。拟议的研究需要与其他项目和核心A进行广泛的互动，并与本PPG的中心主题相匹配。

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.