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: 7033144
• 负责人: JAMES T STULL
• 金额: $ 39万
• 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/7033144
• 关键词: calcium flux; calmodulin dependent protein kinase; cardiac myocytes; cardiogenesis; echocardiography; enzyme activity; fluorescence resonance energy transfer; gene expression; genetically modified animals; heart catheterization; heart contraction; heart enlargement; laboratory mouse; myofibrils; myosin light chain kinase; myosins; newborn animals; phosphorylation; protein isoforms; protein structure function; sarcomeres; smooth muscle

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）：心肌收缩是肌球蛋白与肌动蛋白在肌节细丝和粗丝蛋白调节下的周期性相互作用的结果。基于从皮肤纤维和细胞培养的结果，已经提出，磷酸化的肌节肌球蛋白的调节轻链（RLC）在增强心室收缩中起作用，而磷酸化的肌节或细胞质肌球蛋白-IIB促进肌节组装。钙/钙调蛋白依赖性肌球蛋白轻链激酶（MLCK）表达和活性的功能变化可能导致心肌病。虽然心肌细胞含有平滑肌MLCK，但尚不清楚这是否是唯一负责RLC磷酸化的激酶。我们建议确定的激酶，磷酸化各自的RLC除了RLC磷酸化在心脏收缩和肌节组装的生理作用。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。通过将具有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，消除平滑肌MLCK的基因。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。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。将在来自新生转基因或基因敲除小鼠的肌细胞中测量响应肥大剂的肌节形成。形态分析将与RLC磷酸化测量相结合。将在来自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。