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）：心肌的收缩是由于肌球蛋白与肌动蛋白在肌肉薄细丝蛋白的调节下的循环相互作用引起的。基于培养中皮肤纤维和细胞的结果，已经提出，肉瘤肌球蛋白的调节光链（RLC）的磷酸化在增强心室收缩方面起作用，而肉瘤或细胞质肌球蛋白-IIB的磷酸化则可以促进肉瘤组装。 Ca2+/钙调蛋白依赖性肌球蛋白轻链激酶（MLCK）的表达和活性的功能变化可能导致心肌病。尽管心肌细胞含有光滑的（SM）肌肉MLCK，但尚不清楚这是唯一负责RLC磷酸化的激酶。我们建议确定除了生理作用的RLC磷酸化和肌节组装的生理作用外，还可以磷酸化相应的RLC。具体目标1。确定心室和心肌细胞中SMMLCK的过表达和敲除的影响。转基因小鼠将用特异性在心肌细胞中表达的MLCK制成。平滑肌MLCK的基因将通过将小鼠与平滑肌MLCK基因和专门在心肌细胞中表达CRE的小鼠横穿小鼠来消融。将鉴定出磷酸化RLC的其他激酶。 RLC磷酸化将在心脏组织以及分离的心肌细胞中进行测量。具体目标2。确定功能后果是否与MLCK同工型表达的变化有关。将评估来自转基因和敲除小鼠的心脏的解剖学特性。包括收缩指数在内的功能性能将通过灌注心脏和体内测量，并通过侵入性和无创测量进行测量。还将评估对导致心脏肥大的压力的反应。特定的目标3。确定肌节组件是否受SMMLCK过表达或敲除的影响。响应肥厚剂的肌节形成将在新生儿转基因或敲除小鼠的肌细胞中测量。形态分析将与RLC磷酸化测量结合使用。肌原纤维结构的发展将在胚胎敲除小鼠的心脏中检查。这些研究的结果将为心脏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。