Modulation of Cardiac Function by Myosin Light Chain Kinases and Phosphatases

项目总结 心肌收缩依赖于肌节中维持收缩的分子马达肌球蛋白 表现部分是通过肌球蛋白调节轻链(RLC)的结构性磷酸化来实现的 通过肌球蛋白轻链激酶(MLCK)和磷酸酶(MLCP)的活性。扩张型心脏 小鼠和人的肌病导致心肌MLCK(CMLCK)表达减少和RLC 磷酸化，而磷酸化增加的动物模型增强了心功能 对心力衰竭的抵抗。尽管众所周知，RLC的磷酸化对正常心脏很重要 令人惊讶的是，人们对调控cMLCK和MLCP活性的信号机制知之甚少。 分别进行了分析。我们建议对这两种酶的心脏特异信号机制进行研究 影响肌球蛋白磷酸化以阐明正常功能的细胞机制以及潜在原因 在心力衰竭中RLC磷酸化降低的可能性。具体目标1：最近发现的cMLCK是 在体内被磷酸化，我们计划确定特定的磷酸化位点在调节cMLCK活性中的作用 使用我开发的cMLCK表达和检测系统来检验cMLCK磷酸化的假设 增强了它的活力。这些研究将扩展到完整的心肌，以确定信号机制 参与cMLCK的磷酸化，包括负责的蛋白激酶。此外，我们还将测试 假设其他形式的心力衰竭涉及RLC磷酸化减少，以确定潜在的共同 信号错乱。特定目的2.确定肌球蛋白靶向和可溶性磷酸酶的作用 介导RLC去磷酸化的活性。使用心脏MYPT2的条件性基因敲除模型 相关的、在成年小鼠中普遍表达的亚单位MYPT1，我们将评估特定基因消融的效果 心脏功能。来自野生型和敲除心脏的完整心肌小梁将被用于 定量测量不同磷酸酶池对维持半最大RLC的贡献 磷酸化。来自MyPT1和MyPT2以及PP1cδ基因敲除小鼠的心肌细胞将被用于 鉴定可溶性磷酸酶的调节亚基。这些研究将检验PP1cδ的假设 与MYPT2的结合和解离可特异性地使RLC去磷酸化，从而深入了解RLC的生理作用 心脏中的肌球蛋白磷酸酶。这些结果也将为未来对异常信号的研究奠定基础 通过对RLC磷酸化的影响导致心肌功能障碍的途径。

PROJECT SUMMARY Cardiac contraction depends on the molecular motor myosin in sarcomeres where maintenance of contractile performance is achieved in part by the constitutive phosphorylation of myosin regulatory light chain (RLC) through the respective activities of myosin light chain kinase (MLCK) and phosphatase (MLCP). Dilated cardiac myopathy in mice and humans results in decreased cardiac MLCK (cMLCK) expression and RLC phosphorylation while animal models with increased phosphorylation have enhanced cardiac performance with resistance to heart failure. Although it is well established that RLC phosphorylation is important for normal cardiac function, surprisingly little is known about signaling mechanisms regulating cMLCK and MLCP activities, respectively. We propose investigations on cardiac-specific signaling mechanisms for these two enzymes that affect myosin phosphorylation to elucidate cellular mechanisms for normal function as well as potential causes of decreased RLC phosphorylation in heart failure. Specific Aim 1: Having recently discovered cMLCK is phosphorylated in vivo, we plan to identify roles of specific phosphorylation sites in regulating cMLCK activity using cMLCK expression and assay systems I developed to test the hypothesis that cMLCK phosphorylation enhances its activity. These studies will be extended to intact cardiac muscle to define signaling mechanisms involved in cMLCK phosphorylation, including responsible protein kinases. Additionally, we will test the hypothesis that other forms of heart failure involve reduced RLC phosphorylation to identify potentially common signaling derangements. Specific Aim 2. Determine the roles of myosin-targeted and soluble phosphatase activities in mediating RLC dephosphorylation. Using conditional knockout models for cardiac MYPT2 and the related, ubiquitously expressed subunit MYPT1 in adult mice, we will assess the effects of specific gene ablation on cardiac function. Intact cardiac muscle trabeculae from wildtype and knockout hearts will be used to quantitatively measure contributions of distinct pools of phosphatases to maintenance of half-maximal RLC phosphorylation. Cardiomyocytes from MYPT1 and MYPT2 as well as PP1cδ knockout mice will be used to identify the regulatory subunit for the soluble phosphatase. These studies will test the hypothesis that PP1cδ bound and unbound to MYPT2 specifically dephosphorylates RLC, providing insights into the physiological role of myosin phosphatases in the heart. These results will also set the stage for future studies on aberrant signaling pathways that cause cardiac muscle dysfunction through effects on RLC phosphorylation.