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: 7760983
• 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 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7760983
• 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.

心肌收缩是由肌球蛋白与肌动蛋白在调节下的循环相互作用引起的 肌节细丝和粗丝蛋白。根据带皮纤维和培养细胞的结果，它具有 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，同时肌节或细胞质肌球蛋白-IIB 磷酸化 促进肌节组装。 Ca2+/钙调蛋白-的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 (sm) MLCK，尚不清楚这是负责 RLC 的唯一激酶 磷酸化。我们建议鉴定除磷酸化相应 RLC 之外的激酶 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. 确定心室肌细胞和心房肌细胞中 smMLCK 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。光滑基因 通过将带有 floxed 平滑肌 MLCK 基因的小鼠与小鼠杂交，肌肉 MLCK 将被消除 Cre 特异地在心肌细胞中表达。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。功能特性 包括收缩指数将通过侵入性和非侵入性方法在灌注心脏和体内进行测量 测量。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。 将测量新生儿肌细胞中对肥大剂反应的肌节形成 转基因或基因敲除小鼠。形态测定分析将与 RLC 磷酸化相结合 测量。将在胚胎敲除心脏中检查肌原纤维结构的发育 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。

项目成果

Cardiac myosin light chain is phosphorylated by Ca2+/calmodulin-dependent and -independent kinase activities.

心肌肌球蛋白轻链通过 Ca2/钙调蛋白依赖性和非依赖性激酶活性进行磷酸化。

• 发表时间: 2016-07-05
• 影响因子: 11.1
• 作者: Chang, Audrey N;Mahajan, Pravin;Knapp, Stefan;Barton, Hannah;Sweeney, H Lee;Kamm, Kristine E;Stull, James T
• 通讯作者: Stull, James T