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）：心肌收缩是肌凝蛋白与肌动蛋白在肌体细丝蛋白和粗丝蛋白的调控下循环相互作用的结果。基于皮肤纤维和培养细胞的结果，已经提出肌球蛋白调控轻链（RLC）的磷酸化在增强心室收缩中起作用，而肌球蛋白或细胞质肌球蛋白iib的磷酸化则促进肌节组装。Ca2+/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肌MLCK，但尚不清楚这是否是唯一负责RLC磷酸化的激酶。除了RLC磷酸化在心脏收缩和肌节组装中的生理作用外，我们还建议确定磷酸化相应RLC的激酶。具体目标确定smMLCK在心室和心房肌细胞过表达和敲除的影响。转基因小鼠将在心肌细胞中特异性表达MLCK。平滑肌MLCK基因将通过小鼠与心肌细胞中特异性表达Cre的小鼠杂交来切除。其他磷酸化RLC的激酶将被确定。RLC磷酸化将在心脏组织和分离的肌细胞中测量。具体目标2。确定功能后果是否与MLCK亚型表达的变化有关。将评估转基因和基因敲除小鼠心脏的解剖特性。包括收缩指数在内的功能特性将在灌注心脏和体内通过侵入性和非侵入性测量来测量。对导致心脏肥厚的应激反应也将被评估。具体目标3。确定smMLCK过表达或敲除是否影响肌节组装。在新生转基因小鼠或基因敲除小鼠的肌细胞中测量增生性药物对肌瘤形成的反应。形态计量分析将与RLC磷酸化测量相结合。将在胚胎敲除小鼠的心脏中检查肌纤维结构的发育。这些研究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。