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: 7564721
• 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/7564721
• 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; 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; Proteins; 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; overexpression; response

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.

心肌收缩是肌球蛋白与肌动蛋白在肌动蛋白调控下循环作用的结果 肌节细丝蛋白和粗丝蛋白。根据皮肤纤维和培养细胞的结果，它有 已经提出，肌球蛋白调节轻链(RLC)的磷酸化在 肌球蛋白-IIB的磷酸化增强心室收缩 促进肌节聚集。钙/钙调蛋白表达和活性的功能变化- 依赖肌球蛋白轻链激酶(MLCK)可导致心肌病。虽然心肌细胞 含有平滑(Sm)肌MLCK，目前尚不清楚这是导致RLC的唯一激酶 磷酸化。我们建议除了确定使各自的RLC磷酸化的激酶之外 RLC磷酸化在心脏收缩和肌节组装中的生理作用。特定目标 1.确定SMMLCK在心室肌细胞和心房肌细胞中的过表达和敲除作用。 将用在心肌细胞中特异表达的MLCK制成转基因小鼠。流畅的基因 肌肉MLCK将通过与小鼠和小鼠杂交来消融 Cre在心肌细胞中的特异性表达。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。具体目标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。功能特性 包括收缩指数在内的指标将通过有创和无创两种方式在灌流心脏和活体心脏中进行测量 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。 将测量新生儿心肌细胞对肥大剂的反应的肌节形成 转基因或基因敲除小鼠。形态计量分析将与RLC磷酸化相结合 测量。肌原纤维结构的发育将从胚胎基因敲除开始在心脏中进行检查 老鼠。这些研究的结果将为生理调节和生物化学提供新的见解。 心肌肌球蛋白轻链磷酸化的功能重要性。