Quantitative analysis of cAMP compartmentation in heart

心脏中 cAMP 区室的定量分析

基本信息

批准号：
8305508
负责人：
Jeffrey J. Saucerman
金额：
$ 32.95万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-01 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8305508
关键词：
Actins Address Adrenergic Agents Adrenergic Receptor Arrhythmia Binding Binding Sites Biological Models Biology Biosensor Buffers CREB1 gene Cardiac Cardiac Myocytes Cause of Death Caveolae Cell Nucleus Cell model Complex Computer Simulation Coupling Cyclic AMP Cyclic AMP-Dependent Protein Kinases Cytoskeleton Cytosol Data Developed Countries Diffuse Diffusion Electrophysiology (science)Elements Experimental Designs Experimental Models Fluorescence Resonance Energy Transfer Future Genetic Transcription Growth Health Heart Heart Diseases Heart Rate Image Ion Channel Kinetics Life Measurement Measures Mediating Membrane Microtubules Modeling Molecular Muscle Cells Myocardial Contraction Nuclear Nuclear Pore Nuclear Protein Pathway interactions Phenotype Phosphorylation Phosphotransferases Physiological Play Prostaglandin Receptor Prostaglandins Protein Isoforms Proteins Public Health Pump Regulation Resolution Role Sarcolemma Second Messenger Systems Shapes Signal Pathway Signal Transduction Specificity Stimulus System Systems Biology Testing Therapeutic Therapeutic Agents Ventricular Work adrenergic analog base cellular imaging design heart cell improved innovation insight mRNA Stability novel novel strategies nucleocytoplasmic transport phospholamban phospholemman potassium exchanger sodium-calcium protein distribution real time model receptor research study response second messenger simulation spatiotemporal therapeutic target tool

项目摘要

DESCRIPTION (provided by applicant): Cyclic AMP is a highly versatile second messenger in the heart, transducing an array of different receptor stimuli into coordinated regulation of cardiac functions including excitation-contraction (EC) coupling and gene transcription. But how cAMP can selectively regulate diverse cardiac functions is an important unanswered question in cardiac biology. This lack of basic understanding limits therapeutic strategies for heart disease aimed at suppressing certain cAMP-responsive phenotypes (e.g. structural remodeling, arrhythmia) while preserving other cAMP-responsive phenotypes (e.g. contractility, heart rate). Compartmentation of cAMP and protein kinase A (PKA) has now been directly visualized in cardiac myocytes, and compartmentation is widely hypothesized to be a fundamental mechanism providing cAMP/PKA specificity. The long term objective of this proposal is to develop a systems level understanding of how molecular mechanisms interact to determine cAMP/PKA compartmentation and selective cAMP/PKA signaling. To address this central question, we will use a unique and innovative combination of systems biology approaches: spatiotemporal systems modeling and real-time imaging of cAMP/PKA biosensors in cultured ventricular myocytes. By developing the first molecularly-detailed model of 2-adrenergic signaling (mediated by cAMP and PKA), and the first combination of mechanistic signaling models with FRET biosensors, we pioneered new integrative approaches for understanding cardiac signaling networks from a systems perspective. By iterating between these mechanistic systems models and newly possible experiments in cultured ventricular myocytes, we will test the overall hypothesis that local cAMP/PKA signals are restricted by cAMP degradation, physical barriers, and buffering, which together help mediate selective PKA activity in cytosol, sarcolemma, caveolae, and nucleus. We will test this hypothesis through 3 Specific Aims. Specific Aim 1 characterizes mechanisms restricting local cAMP signals by imaging waves of cAMP diffusion at high spatial and temporal resolution with FRET biosensors and spatially explicit modeling. Specific Aim 2 targets cAMP and PKA FRET biosensors specifically to caveolae, to provide the first direct measurements of local cAMP/PKA signals in this compartment. Finally, Specific Aim 3 examines mechanisms determining how nuclear PKA pathways regulate gene transcription independently of contractile function. Together, these aims will unify our understanding of how cAMP/PKA compartmentation mechanisms selectively coordinate contractility and transcription in response to diverse receptor stimuli. This work reflects a necessary first step towards quantitatively understanding selective regulation of cAMP signaling pathways in the heart. Heart disease is the leading cause of death in the U.S. and many other developed countries. Indeed, the insights provided by this work will aid future efforts towards selectively targeting therapeutics to cardiac disease mechanisms, ultimately improving public health in the U.S. and abroad. PUBLIC HEALTH RELEVANCE: We propose to combine computer modeling and heart cell imaging to identify how cyclic AMP selectively regulates heart contraction vs. heart growth. We expect that this work will aid future efforts towards selectively targeting therapeutic agents towards cardiac disease mechanisms.

描述（由申请人提供）：环 AMP 是心脏中高度通用的第二信使，可将一系列不同的受体刺激转变成心脏功能的协调调节，包括兴奋-收缩 (EC) 耦合和基因转录。但cAMP如何选择性地调节不同的心脏功能是心脏生物学中一个尚未解答的重要问题。这种基本认识的缺乏限制了旨在抑制某些 cAMP 反应表型（例如结构重塑、心律失常）同时保留其他 cAMP 反应表型（例如收缩性、心率）的心脏病治疗策略。现在，cAMP 和蛋白激酶 A (PKA) 的区室在心肌细胞中已被直接观察到，并且区室被广泛假设为提供 cAMP/PKA 特异性的基本机制。该提案的长期目标是在系统水平上了解分子机制如何相互作用以确定 cAMP/PKA 区室和选择性 cAMP/PKA 信号传导。为了解决这个核心问题，我们将使用系统生物学方法的独特和创新组合：时空系统建模和培养心室肌细胞中 cAMP/PKA 生物传感器的实时成像。通过开发第一个 2-肾上腺素信号传导（由 cAMP 和 PKA 介导）的分子详细模型，以及首次将机械信号传导模型与 FRET 生物传感器相结合，我们开创了从系统角度理解心脏信号传导网络的新综合方法。通过在这些机制系统模型和培养的心室肌细胞中新可能的实验之间进行迭代，我们将测试总体假设，即局部 cAMP/PKA 信号受到 cAMP 降解、物理屏障和缓冲的限制，这些信号共同帮助介导细胞质中的选择性 PKA 活性，肌膜、小窝和细胞核。我们将通过 3 个具体目标来检验这一假设。具体目标 1 通过使用 FRET 生物传感器和空间显式建模以高空间和时间分辨率对 cAMP 扩散波进行成像来表征限制局部 cAMP 信号的机制。 Specific Aim 2 针对小窝的 cAMP 和 PKA FRET 生物传感器，首次直接测量该隔室中的局部 cAMP/PKA 信号。最后，具体目标 3 检查了决定核 PKA 通路如何独立于收缩功能调节基因转录的机制。总之，这些目标将统一我们对 cAMP/PKA 区室机制如何选择性协调收缩性和转录以响应不同受体刺激的理解。这项工作反映了定量理解心脏中 cAMP 信号通路选择性调节的必要第一步。心脏病是美国和许多其他发达国家的主要原因。事实上，这项工作提供的见解将有助于未来选择性地针对心脏病机制进行治疗，最终改善美国和国外的公共卫生。公共健康相关性：我们建议结合计算机建模和心脏细胞成像来确定环 AMP 如何选择性调节心脏收缩与心脏生长。我们期望这项工作将有助于未来选择性地针对心脏病机制的治疗药物的努力。