Roles of Myosin Light Chain Kinases in the Heart

肌球蛋白轻链激酶在心脏中的作用

• 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
• 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

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(申请人提供)：心肌收缩是肌球蛋白和肌动蛋白在肌节细丝蛋白和粗丝蛋白的调节下循环作用的结果。根据皮肤纤维和细胞培养的结果，已经提出了肌节肌球蛋白调节轻链(RLC)的磷酸化在促进心室收缩中的作用，而肌节肌球蛋白-IIB的磷酸化促进肌节的组装。钙/钙调素依赖性肌球蛋白轻链激酶(MLCK)表达和活性的功能改变可能导致心肌病。尽管心肌细胞含有平滑(Sm)肌MLCK，但目前尚不清楚这是否是RLC磷酸化的唯一途径。除了RLC磷酸化在心脏收缩和肌节组装中的生理作用外，我们还建议确定使各自的RLC磷酸化的激酶。具体目的1.确定smMLCK在心室肌和心房肌细胞中过表达和敲除的作用。将用在心肌细胞中特异表达的MLCK制成转基因小鼠。通过将平滑肌MLCK基因与在心肌细胞中特异表达CRE的小鼠杂交，将去除平滑肌MLCK基因。其他使RLC磷酸化的激酶将被鉴定出来。将在心脏组织和分离的心肌细胞中测量RLC的磷酸化。具体目的2.确定功能后果是否与MLCK亚型表达的变化有关。我们将评估转基因和基因敲除小鼠心脏的解剖学特性。包括收缩指数在内的功能特性将通过有创和非侵入性测量在灌流心脏和活体心脏中进行测量。对导致心肌肥大的应激反应也将进行评估。特定目的3.确定肌节组装是否受smMLCK过表达或敲除的影响。将在新生转基因或基因敲除小鼠的心肌细胞中测量对肥大剂反应的肌节形成。形态计量分析将与RLC磷酸化测量相结合。肌原纤维结构的发育将在胚胎基因敲除小鼠的心脏中进行检查。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。