Quantitative analysis of cAMP compartmentation in heart

描述(申请人提供)：cAMP是心脏中的一种高度通用的第二信使，将一系列不同的受体刺激转化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cAMP如何选择性地调节多种心脏功能是心脏生物学中一个重要的未解问题。这种基础认识的缺乏限制了心脏病的治疗策略，这些治疗策略旨在抑制某些cAMP反应表型(例如结构重构、心律失常)，同时保留其他cAMP反应表型(例如收缩能力、心率)。目前，cAMP和蛋白激酶A(PKA)的区隔作用已经在心肌细胞中被直接显示，并且区隔作用被广泛认为是提供cAMP/PKA特异性的基本机制。这一建议的长期目标是发展一个系统水平的理解，了解如何相互作用的分子机制，以确定cAMP/PKA区隔和选择性的cAMP/PKA信号。为了解决这个中心问题，我们将使用一种独特的和创新的系统生物学方法的组合：时空系统建模和培养的心室肌细胞cAMP/PKA生物传感器的实时成像。通过发展第一个分子细节的2-肾上腺素能信号模型(由cAMP和PKA介导)，以及第一个机械信号模型与FRET生物传感器的结合，我们开创了从系统角度理解心脏信号网络的新的综合方法。通过在这些机械系统模型和新可能的培养心肌细胞实验之间的迭代，我们将检验局部cAMP/PKA信号受cAMP降解、物理屏障和缓冲限制的总体假设，这些因素共同帮助介导胞浆、肌膜、小窝和细胞核中的选择性PKA活性。我们将通过三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在高空间和时间分辨率成像cAMP扩散波来表征限制局部cAMP信号的机制。特异性目标2以cAMP和PKA FRET生物传感器为靶标，专门针对小窝，以提供该隔室中局部cAMP/PKA信号的第一次直接测量。最后，《特殊目的3》研究了决定核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区隔机制如何选择性地协调收缩和转录以响应不同受体刺激的理解。这项工作反映了定量了解cAMP信号通路在心脏中的选择性调节的必要的第一步。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有选择地针对心脏疾病机制进行治疗的努力，最终改善美国和国外的公共健康。

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.