Roles of Myosin Light Chain Kinases in the Heart

心肌收缩是肌球蛋白与肌动蛋白在肌动蛋白的调节下循环相互作用的结果。 肌节薄丝和粗丝蛋白。根据皮肤纤维和培养细胞的结果， 已经提出肌节肌球蛋白的调节轻链（RLC）的磷酸化在以下方面起作用： 增强心室收缩，同时肌节或细胞质肌球蛋白-IIB磷酸化 促进肌节组装。Ca 2 +/钙调素-受体表达和活性的功能变化 依赖性肌球蛋白轻链激酶（MLCK）可能导致心肌病。虽然心肌细胞 含有平滑肌MLCK，但尚不清楚这是负责RLC的唯一激酶 磷酸化我们建议确定除了磷酸化各自RLC的激酶， RLC磷酸化在心脏收缩和肌节组装中的生理作用。具体目标 1.确定心室和心房肌细胞中smMLCK的过表达和敲除的影响。 将用在心肌细胞中特异性表达的MLCK制备转基因小鼠。光滑的基因 将通过将具有floxed平滑肌MLCK基因的小鼠与具有floxed平滑肌MLCK基因的小鼠杂交来消除肌肉MLCK， 在心肌细胞中特异性表达Cre。将鉴定磷酸化RLC的其他激酶。 将在心脏组织以及分离的肌细胞中测量RLC磷酸化。具体目标2。 确定功能后果是否与MLCK亚型表达的变化相关。 将评估转基因和基因敲除小鼠心脏的解剖学特性。功能特性 包括收缩指数将在灌注心脏和体内通过侵入性和非侵入性测量。 测量.还将评价对导致心脏肥大的应激的反应。 具体目标3。确定肌节组装是否受到smMLCK过表达或敲除的影响。 将在新生儿的肌细胞中测量肌节形成对肥大剂的反应 转基因或敲除小鼠。形态计量学分析将与RLC磷酸化结合 测量.将在胚胎敲除的心脏中检查肌原纤维结构的发育 小鼠这些研究的结果将为生理调节和 心肌肌球蛋白轻链磷酸化的功能重要性。

Contraction of heart muscle results from the cyclic interaction of myosin with actin under the regulation of sarcomeric thin and thick filament proteins. Based on results from skinned fibers and cells in culture it has been proposed that phosphorylation of the regulatory light chain (RLC) of sarcomeric myosin plays a role in enhancing ventricular contraction while phosphorylation of either sarcomeric or cytoplasmic myosin-IIB promotes sarcomere assembly. Functional changes in the expression and activity of Ca2+/calmodulin- dependent myosin light chain kinase (MLCK) may lead to cardiomyopathy. Although cardiac myocytes contain smooth (sm) muscle MLCK, it is not clear that this is the only kinase responsible for RLC phosphorylations. We propose to identify the kinase that phosphorylates the respective RLCs in addition to physiological roles for RLC phosphorylations in cardiac contraction and sarcomere assembly. Specific Aim 1. Determine effects of overexpression and knockout of smMLCK in ventricular and atrial myocytes. Transgenic mice will be made with MLCK expressed specifically in cardiac myocytes. The gene for smooth muscle MLCK will be ablated by crossing mice with the floxed smooth muscle MLCK gene and mice expressing Cre specifically in cardiac myocytes. Other kinases that phosphorylate RLC will be identified. RLC phosphorylations will be measured in cardiac tissues as well as isolated myocytes. Specific Aim 2. Determine if functional consequences are associated with changes in expression of MLCK isoforms. Anatomical properties of hearts from transgenic and knockout mice will be assessed. Functional properties including contractile indices will be measured in perfused hearts and in vivo by invasive and noninvasive measurements. Responsiveness to stresses that lead to cardiac hypertrophy will also be evaluated. Specific Aim 3. Determine if sarcomere assembly is affected by overexpression or knockout of smMLCK. Sarcomere formation in response to hypertrophic agents will be measured in myocytes from newborn transgenic or knockout mice. Morphometric analyses will be combined with RLC phosphorylation measurements. Development of myofibrillar structure will be examined in hearts from embryonic knockout mice. Results from these studies will provide novel biochemical insights into the physiological regulation and functional importance of cardiac myosin light chain phosphorylation.