Modulation of Cardiac Function by Myosin Light Chain Kinases and Phosphatases

肌球蛋白轻链激酶和磷酸酶对心脏功能的调节

• 批准号: 10609853
• 负责人: Audrey N Chang
• 金额: $ 40.5万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10609853
• 关键词: Ablation; Acute; Adult; Affect; Animal Model; Binding; Biochemical; Biological Assay; Cardiac; Cardiac Myocytes; Cardiac Myosins; Cardiomyopathies; Catalytic Domain; Cause of Death; Chemosensitization; DNA Sequence Alteration; Dilated Cardiomyopathy; Dobutamine; Enzymes; Family member; Functional disorder; Future; Genes; Genetic; Goals; Heart; Heart Abnormalities; Heart Diseases; Heart Transplantation; Heart failure; Human; Hypertrophy; Infusion procedures; Investigation; Knock-out; Knockout Mice; Light; Maintenance; Measures; Mediating; Modeling; Molecular Motors; Mus; Muscle; Muscle function; Myocardium; Myopathy; Myosin ATPase; Myosin Light Chain Kinase; Myosin Regulatory Light Chains; Patients; Performance; Pharmacotherapy; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Prevalence; Property; Propranolol; Protein Dephosphorylation; Protein Kinase; Proteins; Resistance; Role; Sarcomeres; Signal Pathway; Signal Transduction; Site; System; Testing; Time; Transcript; cardiac muscle disease; conditional knockout; heart function; improved; in vivo; inorganic phosphate; insight; myosin phosphatase; overexpression; pharmacologic; therapeutic target

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.

项目摘要 心脏收缩依赖于肌节中的分子运动肌球蛋白， 肌球蛋白调节轻链（RLC）的组成性磷酸化部分地实现了性能 通过肌球蛋白轻链激酶（MLCK）和磷酸酶（MLCP）的各自活性。心脏扩张 小鼠和人的肌病导致心脏MLCK（cMLCK）表达和RLC降低 尽管具有增加的磷酸化的动物模型具有增强的心脏性能， 抵抗心力衰竭。尽管已经确定RLC磷酸化对正常心脏的重要性， 功能，令人惊讶的是，对调节cMLCK和MLCP活性的信号传导机制知之甚少， 分别我们建议对这两种酶的心脏特异性信号传导机制进行研究， 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 RLC磷酸化水平降低。具体目标1：最近发现cMLCK是 在体内磷酸化，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表达和测定系统来测试cMLCK磷酸化 增强其活性。这些研究将扩展到完整的心肌，以确定信号机制 参与cMLCK磷酸化，包括负责蛋白激酶。此外，我们将测试 假设其他形式的心力衰竭涉及降低的RLC磷酸化，以确定潜在的共同 信号紊乱具体目标2。确定肌球蛋白靶向磷酸酶和可溶性磷酸酶的作用 介导RLC去磷酸化的活性。使用心脏MYPT 2的条件性敲除模型和 相关的，在成年小鼠中普遍表达的亚基MYPT1，我们将评估特异性基因切除的效果 对心脏功能的影响来自野生型和敲除心脏的完整心肌小梁将用于 定量测量不同的磷酸酶池对维持半最大RLC的贡献 磷酸化来自MYPT 1和MYPT 2以及PP1cδ敲除小鼠的心肌细胞将用于 鉴定可溶性磷酸酶的调节亚基。这些研究将检验PP1cδ 结合和未结合MYPT2特异性去磷酸化RLC，提供了对生理作用的见解 心肌肌球蛋白磷酸酶。这些结果也将为未来异常信号的研究奠定基础 通过影响RLC磷酸化导致心肌功能障碍的途径。