Quantitative analysis of cAMP compartmentation in heart

描述（由申请人提供）：环AMP是心脏中的高度通用的第二信使，将一系列不同的受体刺激转导为心脏功能的协调调节，包括兴奋-收缩（EC）偶联和基因转录。但是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重要的未回答的问题。这种缺乏基本理解限制了旨在抑制某些cAMP响应表型（例如结构重塑、心律失常）同时保留其他cAMP响应表型（例如收缩性、心率）的心脏病治疗策略。cAMP和蛋白激酶A（PKA）的区室化现在已经在心肌细胞中直接可视化，并且区室化被广泛假设为提供cAMP/PKA特异性的基本机制。本提案的长期目标是发展一个系统水平的理解，分子机制如何相互作用，以确定cAMP/PKA区室化和选择性cAMP/PKA信号转导。为了解决这个核心问题，我们将使用一个独特的和创新的组合系统生物学方法：时空系统建模和实时成像的cAMP/PKA生物传感器在培养的心室肌细胞。通过开发第一个分子详细的2-肾上腺素能信号传导模型（由cAMP和PKA介导），以及第一个机械信号传导模型与FRET生物传感器的组合，我们开创了从系统角度理解心脏信号传导网络的新的综合方法。通过迭代这些机制系统模型和新的可能的实验在培养的心室肌细胞，我们将测试的整体假设，当地的cAMP/PKA信号受到限制的cAMP降解，物理屏障，和缓冲，共同帮助介导选择性PKA活性在胞质溶胶，肌膜，小窝，和细胞核。我们将通过三个具体目标来验证这一假设。具体目标1的特点是机制限制本地cAMP信号成像波cAMP扩散在高空间和时间分辨率与FRET生物传感器和空间显式建模。特异性目标2将cAMP和PKA FRET生物传感器特异性靶向小窝，以提供该隔室中局部cAMP/PKA信号的第一次直接测量。最后，具体目标3研究机制，决定核PKA途径如何调节基因转录独立收缩功能。总之，这些目标将统一我们对cAMP/PKA区室化机制如何选择性地协调收缩性和转录以响应不同受体刺激的理解。这项工作反映了必要的第一步定量了解选择性调节cAMP信号通路在心脏。心脏病是美国和许多其他发达国家的主要死亡原因。事实上，这项工作提供的见解将有助于未来的努力，以选择性地靶向治疗心脏病机制，最终改善美国和国外的公共卫生。公共卫生相关性：我们建议将联合收割机计算机建模和心脏细胞成像相结合，以确定环磷酸腺苷如何选择性地调节心脏收缩与心脏生长。我们希望这项工作将有助于未来的努力，选择性靶向治疗药物对心脏疾病的机制。

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.