Quantitative analysis of cAMP compartmentation in heart

心脏中 cAMP 区室的定量分析

• 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
• 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

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）：循环AMP是心脏中高度用途的第二使者，将一系列不同的受体刺激阵列转换为对心脏功能的协调调节，包括激发诱导（EC）耦合和基因转录。但是，CAMP如何有选择地调节多样化的心脏功能是心脏生物学中重要的未解决的问题。缺乏基本理解限制了旨在抑制某些营地反应性表型（例如结构重塑，心律不齐）的心脏病的治疗策略，同时保留其他营地反应性表型（例如，收缩力，心率）。 CAMP和蛋白激酶A（PKA）的隔室已直接在心肌细胞中直接可视化，并且广泛认为隔室是提供CAMP/PKA特异性的基本机制。该提案的长期目标是对分子机制如何相互作用以确定CAMP/PKA隔室和选择性CAMP/PKA信号的相互作用来建立系统级别的理解。为了解决这个中心问题，我们将使用系统生物学方法的独特而创新的组合：在培养的心室心肌细胞中营地/PKA生物传感器的时空系统建模和实时成像。通过开发2-肾上腺素信号传导（由CAMP和PKA介导的）的第一个分子检测模型，以及机械信号模型与FRET生物传感器的首次组合，我们开创了从系统角度来理解心脏信号网络的新的整合方法。通过在这些机械系统模型和在培养的心室肌细胞中进行新的可能的实验之间进行迭代，我们将测试总体假设，即当地营地/PKA信号受营地退化，物理屏障和缓冲的限制，这些假设共同介导了选择性PKA活性在细胞质，肌肉溶质，Sarcolemma，cavelolea和unucleus和unucleus和unucleus和unucleus中。我们将通过3个特定目标检验这一假设。特定的目标1表征了通过在高空间和时间分辨率的cAMP扩散的图像中限制了本地cAMP信号的机制，并具有FRET生物传感器和空间显式建模。特定的目标2针对CAMP和PKA FRET生物传感器，专门针对Caveolae，以在此隔间中对本地营地/PKA信号进行了首次直接测量。最后，具体目标3研究了确定核PKA途径如何独立于收缩功能调节基因转录的机制。共同，这些目标将统一我们对CAMP/PKA隔室如何选择性地协调收缩性和转录的理解，以响应多种受体刺激。这项工作反映了朝着定量理解心脏中cAMP信号通路的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死亡的主要原因。实际上，这项工作提供的见解将有助于未来的努力，以选择性地靶向心脏病机制，最终改善美国和国外的公共卫生。公共卫生相关性：我们建议将计算机建模和心细胞成像结合起来，以确定环状AMP如何有选择地调节心脏收缩与心脏的生长。我们预计这项工作将有助于未来的努力，以选择性地针对心脏病机制的治疗剂。