Roles of Myosin Light Chain Kinases in the Heart

肌球蛋白轻链激酶在心脏中的作用

基本信息

批准号：
7171824
负责人：
JAMES T STULL
金额：
$ 38.11万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-02-01 至 2011-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7171824
关键词：
Ablation Actins Affect Biochemical Biosensor Birth Breeding Calcium Calmodulin Cardiac Cardiac Myocytes Cardiac Myosins Cardiomyopathies Catheterization Cells Congestive Heart Failure Consensus Data Defect Development Echocardiography Embryonic Heart Fiber Fluorescence Resonance Energy Transfer Genes Heart Heart Atrium Heart Hypertrophy Histology Invasive Investigation Knock-out Knockout Mice Lead Left Length Light Measurement Measures Mus Muscle Cells Myocardium Myosin ATPase Myosin Light Chain Kinase Myosin Light Chains Myosin Type II Newborn Infant Nonmuscle Myosin Type IIB Performance Phosphorylation Phosphotransferases Physiological Play Procedures Property Protein Isoforms Protein Overexpression Proteins Rate Regulation Research Role Sarcomeres Skeletal Muscle Myosins Skin Stress Structure Testing Thick Filament Tissues Transgenic Mice Transgenic Organisms Transmission Electron Microscopy Ventricular Weight base fiber cell gain of function in vivo indexing insight knockout animal knockout gene novel response size

项目摘要

DESCRIPTION (provided by applicant): 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 phosphorylation. 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.

描述（由申请人提供）：心肌的收缩是由肌球蛋白与肌动蛋白在肌节细丝蛋白和粗丝蛋白的调节下的循环相互作用引起的。根据带皮纤维和培养细胞的结果，有人提出，肌节肌球蛋白调节轻链 (RLC) 的磷酸化在增强心室收缩中发挥作用，而肌节或细胞质肌球蛋白-IIB 的磷酸化则促进肌节组装。 Ca2+/钙调蛋白依赖性肌球蛋白轻链激酶 (MLCK) 的表达和活性的功能性变化可能导致心肌病。尽管心肌细胞含有平滑肌 (sm) MLCK，但尚不清楚这是负责 RLC 磷酸化的唯一激酶。除了 RLC 磷酸化在心脏收缩和肌节组装中的生理作用之外，我们还建议鉴定磷酸化相应 RLC 的激酶。具体目标 1. 确定 smMLCK 在心室和心房肌细胞中过表达和敲除的影响。将使用在心肌细胞中特异表达的 MLCK 来培育转基因小鼠。通过将带有 floxed 平滑肌 MLCK 基因的小鼠与在心肌细胞中特异性表达 Cre 的小鼠杂交，可以消除平滑肌 MLCK 基因。其他磷酸化 RLC 的激酶也将被鉴定。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 同工型表达的变化相关。将评估转基因和基因敲除小鼠心脏的解剖学特性。将通过侵入性和非侵入性测量在灌注心脏和体内测量包括收缩指数在内的功能特性。还将评估对导致心脏肥大的压力的反应。具体目标 3. 确定肌节组装是否受到 smMLCK 过度表达或敲除的影响。将在新生转基因或基因敲除小鼠的肌细胞中测量对肥大剂反应的肌节形成。形态测定分析将与 RLC 磷酸化测量相结合。将在胚胎敲除小鼠的心脏中检查肌原纤维结构的发育。这些研究的结果将为心肌肌球蛋白轻链磷酸化的生理调节和功能重要性提供新的生化见解。