cAMP Compartmentation in Cardiac Myocytes

CAMP信号通路在心肌细胞许多不同方面的调控中起着关键作用 功能，包括基因转录、细胞代谢和兴奋-收缩偶联。然而，不是 所有刺激cAMP产生的G蛋白偶联受体都会产生相同的反应。亚细胞 CAMP的区隔对于解释不同的受体如何利用相同的扩散性是必不可少的。 第二信使引起独特的功能反应。此外，营地的中断 分区与各种疾病状态有关，包括心肌肥大、心力衰竭、 还有心律失常。然而，对营地划分的机制有一个完整的了解 仍然是个谜。大多数工作都集中在磷酸二酯酶(PDE)的作用上，它可以分解 营地，通常被认为是功能障碍或活动汇，定义了不同 信令域。然而，一些研究表明，仅有PDE活动是不够的。这个 结果表明，除了PDE活性，独特的受体依赖反应只能是 解释了细胞内cAMP的移动是否受到其他机制的限制。使用 先进的新方法，我们已经直接测量了完整的cAMP的扩散系数 并发现它的移动速度确实比之前想象的要慢得多。此外， 我们的初步数据确定了两个对解释这一行为至关重要的因素。第一个是缓冲 蛋白激酶A(PKA)通过蛋白激酶锚定蛋白(AKAPs)固定的cAMP。第二个是 亚细胞受限空间。在这项提案中，我们将解决以下具体问题：1)是否 线粒体外膜PKA的缓冲作用对cAMP的贡献 车厢分隔？2)与二叉裂相关的受限空间是否有助于cAMP 车厢分隔。为了回答这些问题，我们将使用分子、生化和 生物物理技术。其中包括栅格图像相关光谱(RICS)，用于直接测量 CAMP扩散、荧光共振能量转移(FRET)生物传感器针对不同 测量cAMP区划的亚细胞位置；以及膜片钳电生理学，钙 荧光分光光度法和心肌细胞缩短法测量分区的cAMP依赖功能 回应。这些问题的答案可能会导致开发出新的停车方法 控制心血管疾病的发展并预防其致命后果。

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.