cAMP Compartmentation in Cardiac Myocytes

心肌细胞中的 cAMP 区室

• 批准号: 10321915
• 负责人: ROBERT D HARVEY
• 金额: $ 36万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-15 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10321915
• 关键词: A kinase anchoring protein; Address; Area; Arrhythmia; Autonomic nervous system; Behavior; Binding Sites; Biochemical; Biosensor; Blood; Buffers; Ca(2+)-Calmodulin Dependent Protein Kinase; Cardiac Myocytes; Cardiovascular Diseases; Cell Volumes; Cell membrane; Cells; Cellular Metabolic Process; Complex; Coupling; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Development; Diffuse; Diffusion; Disease; Electrophysiology (science); Exercise; Fluorescence Resonance Energy Transfer; Fluorometry; G-Protein-Coupled Receptors; Genetic Transcription; Goals; Health; Heart; Heart Hypertrophy; Heart failure; Holoenzymes; Hormones; Image; Immobilization; Individual; Lead; Link; Location; Measures; Mediating; Membrane; Membrane Microdomains; Microfilaments; Mitochondria; Molecular; Movement; Muscle Cells; Neurotransmitters; Outer Mitochondrial Membrane; Peripheral; Phosphotransferases; Play; Production; Protein Isoforms; Proteins; Role; Sarcoplasmic Reticulum; Second Messenger Systems; Signal Pathway; Signal Transduction; Site; Spectrum Analysis; Stress; Study models; Subcellular Spaces; Techniques; Testing; Work; base; beta-adrenergic receptor; biophysical techniques; experimental study; heart cell; heart electrical activity; heart function; junctophilin; novel strategies; patch clamp; phosphoric diester hydrolase; prevent; receptor; response; segregation; sudden cardiac death; theories; tool

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.

营地信号通路在调节心肌细胞的许多不同方面起着至关重要的作用 功能，包括基因转录，细胞代谢和兴奋 - 收缩耦合。但是，不是 刺激cAMP产生的所有G蛋白偶联受体产生相同的反应。亚细胞 cAMP的隔室对于解释不同受体如何利用相同的扩散至关重要 第二使者引起独特的功能响应。此外，营地的破坏 隔室已与各种疾病状态有关，包括心脏肥大，心力衰竭， 和心律不齐。然而，促成营地隔间的机制的完整图片 仍然是一个谜。大多数工作都集中在磷酸二酯酶（PDES）的作用上 营地，通常被认为是功能性障碍或定义不同的主动水槽 信号域。但是，许多研究表明，仅PDE活性是不够的。这 结果表明，除了PDE活性外，独特的受体依赖性反应只能是 解释了细胞内营地的运动是否受其他机制的限制。使用 精致的新方法，我们直接测量了CAMP的扩散系数 心肌细胞发现它的确比以前想象的要慢得多。此外， 我们的初步数据已经确定了对解释这种行为至关重要的两个因素。第一个是缓冲 蛋白激酶A（PKA）扎营，被激酶锚定蛋白（AKAP）固定。第二个是 亚细胞限制空间。在此提案中，我们将解决以下特定问题：1） PKA锚定在线粒体的外膜上的缓冲有助于营地 隔间？ 2）与二元裂口相关的受限空间有助于营地 隔室。为了回答这些问题，我们将结合分子，生化和 生物物理技术。这些包括栅格图像相关光谱（RIC）直接测量 CAMP扩散，荧光共振能量转移（FRET）的生物传感器针对不同的生物传感器 亚细胞位置以测量营地隔室；和斑块夹电生理学，CA2+ 荧光测定法和肌细胞缩短以测量隔室化cAMP依赖性功能 回答。这些问题的答案可能导致制定新颖方法的制定方法 心血管疾病的进展并预防致命后果。