Ca-Calmodulin Activated Phosphorylation Signaling Pathways in the Heart

钙钙调蛋白激活心脏磷酸化信号通路

• 批准号: 8415645
• 负责人: Courtney Blake Nichols
• 金额: $ 5.22万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8415645
• 关键词: Action Potentials; Adult; Affinity; Agonist; Animal Model; Arrhythmia; Binding; Ca(2+)-Calmodulin Dependent Protein Kinase; Calcineurin; Calcineurin Pathway; Calcium; Calmodulin; Calmodulin-Binding Proteins; Cardiac; Cardiac Myocytes; Cardiac Output; Cells; Cleaved cell; Coupling; Critical Pathways; Cytosol; Endothelin-1; Fellowship; Fluorescence; Fluorescence Resonance Energy Transfer; Frequencies; G-Protein-Coupled Receptors; Growth and Development function; Heart; Heart Hypertrophy; Heart failure; Hormonal; Human; Imaging Device; Investigation; Ion Channel; Kinetics; Life; Location; Measures; Mechanics; Mediating; Membrane; Memory; Modeling; Molecular; Monitor; Mus; Muscle Cells; Nodal; Nuclear; Pathology; Peptide Hydrolases; Phosphorylation; Physiological; Play; Process; Proteins; Pump; Recovery; Reporter; Role; Sarcoplasmic Reticulum; Second Messenger Systems; Signal Pathway; Signal Transduction; Signaling Molecule; Stimulus; Techniques; Testing; Time; Transcriptional Regulation; Transgenic Animals; Transgenic Mice; Translating; Ventricular; base; calmodulin-dependent protein kinase II; career development; cellular imaging; heart cell; mutant; new therapeutic target; novel; oxidation; prevent; relating to nervous system; response; second messenger; sensor; therapeutic target; tool

DESCRIPTION (provided by applicant): Ca2+ is the principal second messenger in translating electrical signals (i.e. action potentials) into mechanical process in the heart (i.e. contractions. This highly orchestrated process of excitation-contraction coupling (ECC) is achieved through a number of transporters, pumps, and ion channels on the sarcolemmal membrane as well as in the sarcoplasmic reticulum (SR), resulting in the Ca2+ waves observed during a beat-to-beat cycle. Modulation of these Ca2+ handling proteins by the ubiquitously expressed Ca2+/calmodulin-dependent protein kinase II (CaMKII) and calcineurin (CaN) have been found to play an increasingly important role in the heart. Cardiomyocytes signaling during ECC is described by the flux in intracellular [Ca2+]i concentration. During the increase in [Ca2+]i Ca2+ bound calmodulin (Ca-CaM) binds to and activates CaMKII and CaN; thereby phosphorylating or dephosphorylating several important Ca2+ handling proteins with multiple functional consequences within the cell. CaMKII and CaN signaling are independent of each other, but can also be antagonistic in the phosphorylation signaling cascade. CaMKII activity and expression were shown to be increased in human cardiac hypertrophy, heart failure, and animal models of heart failure. Inhibiting CaMKII activity in animal models of heart failure reduces or prevents pathology. The inhibition of CaN has demonstrated beneficial effect in animal models of heart failure; however the signaling mechanisms are not understood and need further investigation. Thus, these Ca-CaM-dependent pathways are critical nodal points and potentially valuable therapeutic targets in the genesis and treatment of heart failure and arrhythmias. The specific aims are focus on localization and translocation (A) and activation dynamics (B) of the Ca-CaM- CaMKII-CaN signaling pathways with the following hypothesizes: (1A) Ca-CaM localization and translocation are induced by different stimuli. (1B) Ca-CaM signaling dynamics differ in key myocyte locations. (2A) CaMKII4B vs. 4C differ in localization and translocation. (2B) CaMKII activation in myocytes differs locally and integrates to cause "memory". (3A) CaN localizes at the Z-line, but translocates with local Ca2+ signals. (3B) Kinetics and location of CaN activation differ from CaMKII in adult cardiomyocytes. The Bers Lab has generated a set of fluorescently tagged proteins to monitor localization and translocation of CaM, CaMKII and CaN. There are also FRET based activity reporters for Ca-CaM, CaMKII and CaN. These constructs will be transfected into isolated adult cardiomyocytes for live cell imaging and the use of FRAP and TIRF will be employed to investigate the dynamics of the signaling pathways. After characterization of the Ca-CaM-CaMKII- CaN pathway in heathly heart cells, cardiomyocytes from heart failure models will be used to investigate pathological changes. This will greatly enhance our understanding of how CaMKII and CaN activity is regulated in the heart by both pathological and physiological signaling pathways. This will help to identify novel signaling cascades and thereby identify new therapeutic targets for the treatment of heart failure.

描述（由申请人提供）：Ca2+是将电信号（即动作电位）转化为心脏机械过程（即收缩）的主要第二信使。这种高度协调的兴奋-收缩耦合（ECC）过程是通过肌膜和肌质网（SR）上的许多转运蛋白、泵和离子通道来实现的， 导致在逐次心跳周期中观察到 Ca2+ 波。人们发现，普遍表达的 Ca2+/钙调蛋白依赖性蛋白激酶 II (CaMKII) 和钙调神经磷酸酶 (CaN) 对这些 Ca2+ 处理蛋白的调节在心脏中发挥着越来越重要的作用。 ECC 期间的心肌细胞信号传导通过细胞内 [Ca2+]i 浓度的变化来描述。期间 [Ca2+]i Ca2+ 结合钙调蛋白 (Ca-CaM) 的增加结合并激活 CaMKII 和 CaN；从而磷酸化或去磷酸化几种重要的 Ca2+ 处理蛋白，在细胞内产生多种功能后果。 CaMKII 和 CaN 信号传导彼此独立，但在磷酸化信号传导级联中也可以是拮抗的。 CaMKII 活性和表达在人类中显示增加 心脏肥大、心力衰竭和心力衰竭动物模型。抑制心力衰竭动物模型中的 CaMKII 活性可减少或预防病理。 CaN 的抑制已在心力衰竭动物模型中显示出有益的效果；然而，信号机制尚不清楚，需要进一步研究。因此，这些 Ca-CaM 依赖性途径是心力衰竭和心力衰竭的发生和治疗中的关键节点和潜在有价值的治疗靶点。 心律失常。具体目标是关注 Ca-CaM-CaMKII-CaN 信号通路的定位和易位 (A) 以及激活动力学 (B)，并提出以下假设：(1A) Ca-CaM 定位和易位是由不同刺激诱导的。 (1B) Ca-CaM 信号传导动力学在关键的心肌细胞位置上有所不同。 (2A) CaMKII4B 与 4C 在定位和易位方面有所不同。 (2B) 肌细胞中的 CaMKII 激活局部不同并整合以引起“记忆”。 (3A) CaN 定位于 Z 线，但随局部 Ca2+ 信号易位。 (3B) 成人心肌细胞中 CaN 激活的动力学和位置与 CaMKII 不同。 Bers 实验室生成了一组荧光标记蛋白来监测 CaM、CaMKII 的定位和易位 和CaN。还有基于 FRET 的 Ca-CaM、CaMKII 和 CaN 活动报告基因。这些构建体将被转染到分离的成体心肌细胞中进行活细胞成像，并且将使用 FRAP 和 TIRF 来研究信号传导途径的动态。在对健康心脏细胞中的 Ca-CaM-CaMKII-CaN 通路进行表征后，心力衰竭模型中的心肌细胞将用于研究病理变化。 这将极大地增强我们对心脏中 CaMKII 和 CaN 活性如何通过病理和生理信号通路进行调节的理解。这将有助于识别新的信号级联，从而确定治疗心力衰竭的新治疗靶点。