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，血管平滑肌收缩性调控的复杂过程，从而导致高血压、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）的收缩性是通过肌球蛋白调节轻链（RLC）的磷酸化开启的，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）和肌球蛋白轻链磷酸酶的相反活性，两者都可以通过上游信号通路调节。我们在项目2中产生了MLCK缺失小鼠，这些小鼠从E15.5开始胚胎致死，一些达到足月，但在此之前，胚胎主动脉或脐带血管显示RLC磷酸化和正常力发育，以响应ca2 +。我们将验证普遍表达的SM mlck分别对平滑肌和心肌的收缩、迁移、细丝和肌节形成以及A404SMC“祖”细胞（项目2，核心A）和转化的心外膜前细胞（项目3，核心A）至关重要的假设；或者，在MLCK缺失的胚胎中，有一种代偿性激酶负责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，在无MLCK胚胎中，冠状血管的形成存在缺陷，因此，我们将用Project 3验证MLCK对心外膜前器官（冠状血管的前体）的心外膜细胞迁移至关重要的假设。细胞迁移和收缩背后的肌球蛋白运动也受到调节，并有助于SM收缩表型。这些机制将探索使用动力学分析与小说

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.