Quantitative analysis of cAMP compartmentation in heart

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如何选择性地调节各种心脏功能是心脏生物学中一个重要的未解之谜。这种基本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（例如结构重塑，心律失常），同时保留其他camp反应表型（例如收缩性，心率）。cAMP和蛋白激酶A （PKA）的区隔作用现在已经在心肌细胞中被直接观察到，并且区隔作用被广泛假设是提供cAMP/PKA特异性的基本机制。本提案的长期目标是发展对分子机制如何相互作用以确定cAMP/PKA区隔和选择性cAMP/PKA信号传导的系统级理解。为了解决这个核心问题，我们将使用一种独特而创新的系统生物学方法组合：在培养的心室肌细胞中对cAMP/PKA生物传感器进行时空系统建模和实时成像。通过建立第一个2-肾上腺素能信号（由cAMP和PKA介导）的分子详细模型，以及第一个将机制信号模型与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网络的新综合方法。通过在这些机制系统模型和在培养心室肌细胞中进行的新可能的实验之间进行迭代，我们将验证局部cAMP/PKA信号受到cAMP降解、物理屏障和缓冲的限制的总体假设，这些因素共同帮助介导细胞质、肌膜、小泡和细胞核中的选择性PKA活性。我们将通过3个具体目标来检验这一假设。特异性目标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下成像cAMP扩散波，表征限制局部cAMP信号的机制。Specific Aim 2将cAMP和PKA FRET生物传感器特异靶向于小泡，从而首次直接测量该小泡中的局部cAMP/PKA信号。最后，Specific Aim 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的第一步。在美国和许多其他发达国家，心脏病是导致死亡的主要原因。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病机制进行治疗，最终改善美国和国外的公众健康。

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.