cAMP Compartmentation in Cardiac Myocytes

cAMP信号通路在调节心肌细胞的许多不同方面起着关键作用 功能，包括基因转录，细胞代谢和兴奋-收缩偶联。但并非 所有刺激cAMP产生的G蛋白偶联受体产生相同的反应。亚细胞 cAMP的区室化对于解释不同的受体如何利用相同的可扩散性是必不可少的。 第二信使以引起独特的功能反应。此外，破坏cAMP 区室化与各种疾病状态有关，包括心脏肥大，心力衰竭， 和心律失常。然而，有助于cAMP区室化的机制的全貌 仍然是个谜大多数工作都集中在磷酸二酯酶（PDE）的作用， cAMP和通常被认为是功能性屏障或活性汇， 信号域然而，许多研究表明，PDE活性本身是不够的。的 结果表明，除了PDE活性，独特的受体依赖性反应只能是 解释了细胞内cAMP的运动是否受到其他机制的限制。使用 先进的新方法，我们直接测量了cAMP在完整的细胞中的扩散系数， 发现它确实比以前认为的移动得慢得多。此外，委员会认为， 我们的初步数据已经确定了两个关键因素来解释这种行为。第一个是缓冲 cAMP通过由A激酶锚定蛋白（AKAP）固定的蛋白激酶A（PKA）。其次是 亚细胞受限空间。在本建议书中，我们将讨论以下具体问题：1）是否 锚定在线粒体外膜上的PKA的缓冲作用有助于cAMP 分隔？和2）与二元分裂相关的受限空间是否有助于cAMP 分隔。为了回答这些问题，我们将结合分子、生物化学和 生物物理技术这些包括光栅图像相关光谱（RICS），以直接测量 cAMP扩散，荧光共振能量转移（FRET）为基础的生物传感器，针对不同的 测量cAMP区室化的亚细胞位置;和膜片钳电生理学，Ca 2 + 荧光测定法和肌细胞缩短来测量区室化cAMP依赖性功能性 应答这些问题的答案可能会导致开发新的方法来停止 心血管疾病的发展和预防致命的后果。

The cAMP signaling pathway plays a critical role in regulating many different aspects of cardiac myocyte function, including gene transcription, cell metabolism, and excitation-contraction coupling. However, not all G-protein coupled receptors that stimulate cAMP production produce the same responses. Subcellular compartmentation of cAMP is essential to explain how different receptors can utilize the same diffusible second messenger to elicit unique functional responses. Furthermore, disruption of cAMP compartmentation has been linked to various disease states, including cardiac hypertrophy, heart failure, and arrhythmias. Yet, a complete picture of the mechanisms contributing to cAMP compartmentation remains a mystery. Most work has focused on the role of phosphodiesterases (PDEs), which breakdown cAMP and are commonly thought to act as either functional barriers or active sinks that define different signaling domains. However, a number of studies indicate that PDE activity alone is not sufficient. The results suggest that, in addition to PDE activity, unique receptor-dependent responses can only be explained if the movement of intracellular cAMP is somehow limited by other mechanisms. Using a sophisticated new approach, we have directly measured the diffusion coefficient of cAMP in intact myocytes and found that it does indeed move dramatically slower than previously thought. Furthermore, our preliminary data have identified two factors critical to explaining this behavior. The first is buffering of cAMP by protein kinase A (PKA) immobilized by A kinase anchoring proteins (AKAPs). The second are subcellular restricted spaces. In this proposal, we will address the following specific questions: 1) Does buffering by PKA anchored to the outer membrane of mitochondria contribute to cAMP compartmentation? and 2) Does the restricted space associated with dyadic clefts contribute to cAMP compartmentation. To answer these questions, we will use a combination of molecular, biochemical, and biophysical techniques. These include raster image correlation spectroscopy (RICS) to directly measure cAMP diffusion, fluorescence resonance energy transfer (FRET)-based biosensors targeted to different subcellular locations to measure cAMP compartmentation; and patch clamp electrophysiology, Ca2+ fluorometry, and myocyte shortening to measure compartmentalized cAMP-dependent functional responses. The answers to these questions could lead to the development of novel approaches to halting the progression of cardiovascular disease and preventing the deadly consequences.