Regulatory Mechanisms in Lymphatic Muscle Contraction

描述(由申请人提供)： 我目前的职业目标是建立高质量研究的声誉，培养心血管和淋巴研究领域的研究生和博士后。我的长期职业是提高对正常和疾病/病理生理条件下淋巴肌肉收缩调节机制的理解水平。淋巴肌表现出强烈的时相收缩，更高的收缩速度和不同的细胞内钙动力学。不幸的是，人们对导致这些独特特征的分子机制知之甚少。在这个方案中，我们将使用现有的收缩蛋白基因敲除模型或过度表达收缩蛋白亚型的转基因模型来检验中心假设：淋巴肌肉收缩装置中粗或细丝蛋白的重塑通过改变钙敏感性和交叉桥激活机制来调节其收缩动力学。其具体目的是：(1)确定SM-B肌球蛋白重链(MHC)在淋巴管时相和紧张性收缩中的功能作用；(2)确定原肌球蛋白在细丝介导的淋巴管收缩中的作用。我们将使用SM-B MHC基因敲除和SM-MHC/Calponin双基因敲除小鼠模型。血管平滑肌α-肌动蛋白启动子将在淋巴肌中表达横纹肌原肌球蛋白。腺病毒siRNA方法将被用来下调淋巴管中平滑肌a-和p-TM基因的表达。我们将从髂管和胸管淋巴管分离/插管血管，以研究淋巴管的收缩特性。将在淋巴管段制剂(包括完整的和去皮的)中进行力和钙的测量，以确定淋巴管的钙敏感性和协同机制。淋巴管的收缩机制知之甚少，这项研究将极大地提高我们对淋巴管功能基础的认识。这些研究进一步推进了我使用小鼠模型进行的训练淋巴研究，这将使我能够开发和使用基因组蛋白质组方法来确定调节淋巴肌肉功能的信号通路。 (摘要结束)

DESCRIPTION (provided by applicant): My immediate career objectives are to develop a reputation for high quality research and to train graduate and postdoctoral students in the cardiovascular and lymphatic research field. My long-term career are to advance the level of understanding the regulatory mechanisms of lymphatic muscle contractility under normal and diseased/pathophysiological conditions. Lymphatic muscle exhibits strong/phasic contractions, much higher shortening velocities and different intracellular calcium dynamics. Unfortunately little information is known about the molecular mechanisms that are responsible for these unique characteristics. In this proposal we will use the available contractile protein gene knock out models or transgenic models with overexpression of contractile protein isoforms to test the central hypothesis remodeling of thick or thin filament proteins in the contractile apparatus of lymphatic muscle modulates its contractile dynamics through altering the calcium sensitivity and crossbridge activation mechanisms. The specific aims are: (1) To determine the functional roles of SM-B myosin heavy chain (MHC) in the phasic and tonic contraction of lymphatics, (2) To define the roles of tropomyosin in the thin filament-mediated contraction of lymphatics. We will use SM-B MHC knockout and SM-MHC/calponin double knockout mouse models. The vascular smooth muscle a-actin promoter will be to express striated muscle tropomyosin in lymphatic muscle. An adenoviral siRNA approach will be used knock down the smooth muscle a- and p-TM gene expression in lymphatics. We will isolated/cannulated vessels from iliac and thoracic duct lymphatics to study the contractile characteristics of lymphatics. Force and calcium measurements will be conducted in the lymphatic segment preparations (both intact and skinned) to determine the calcium sensitivity and cooperativity mechanisms of lymphatics. The contractile mechanisms of lymphatics are poorly understood and this study will significantly advance our knowledge of the basis for the lymphatic vessel function. These studies further advance my training lymphatic research using mouse models, which would allow me to develop and use genomic proteomic approaches to determine the signaling pathways that regulate lymphatic muscle function. (End of Abstract)