Role of mitochondria-associated spaces in the regulation of compartmentation of cAMP signaling

项目摘要 不同的G蛋白偶联受体在细胞内引起不同的功能反应，即使它们使用的是 常见的可扩散第二信使cAMP。例如，当刺激β-肾上腺素能受体或 E型前列腺素受体导致cAMP的产生，只有β-肾上腺素能受体调节心肌细胞 收缩性细胞区分同一细胞中产生的cAMP的能力只能解释为， 不同受体的结合产生不同的cAMP受体特异性库。然而，底层 负责产生区室化cAMP的机制还不完全清楚。 区室化cAMP信号传导调节心脏收缩力，因此对于心脏的正常功能是必不可少的。 心脏与此相一致，cAMP区室化的失调与几个 心血管疾病，包括心律失常、肥大和心力衰竭。大多数先前的研究 研究人员主要关注磷酸二酯酶（分解cAMP的酶）的活性来解释cAMP 分隔然而，一些数学研究已经预测，PDE活性本身并不重要。 足够了。这些研究表明，cAMP的流动性必须比自由扩散慢，以防止 cAMP到达非特异性靶蛋白。我们最近已经证明，细胞内流动性 cAMP的释放明显地受到由α-相关蛋白激酶A介导的缓冲作用的阻碍。一项新 计算研究已经预测，除了cAMP的缓慢扩散之外，解剖学上限制的空间 是阻碍cAMP运动的关键。在心肌细胞中，线粒体占细胞的30 通过与肌浆网的相互作用， 细胞骨架蛋白本提案的总体目标是探索与紧凑空间相关的概念， 线粒体调节cAMP区室化。线粒体与肌浆网的连接 通过蛋白质线粒体融合蛋白-2（MFN 2）、葡萄糖调节蛋白75（GRP 75）和磷酸弗林酸性簇 分选蛋白2（PACS 2）在这些细胞器之间产生紧密的空间。在本研究的第一个目的中，我们 将检验线粒体和肌浆之间解剖学上限制的空间 网状纤维阻碍cAMP运动并有助于cAMP区室化。在心肌细胞中，线粒体 这种排列受微管和肌肉LIM蛋白（MLP）的调节。微管或MLP破坏 导致线粒体解体并改变线粒体形态，从而改变细胞质 与线粒体有关的空间。因此，在第二个目的中，我们假设cAMP区室化 在微管破坏的细胞中线粒体紊乱后受到阻碍。为了检验这些假设，我们 采用多管齐下和互补的方法来研究cAMP区室化。使用各种 先进的技术，我们将测量cAMP的流动性，在特定的细胞内cAMP水平的变化， 位置，Ca 2+通道电流和细胞内Ca 2+瞬变的变化，以及功能性 反应，如细胞缩短后刺激个别受体。本提案的目的是 阐明负责促进cAMP区室化的基本机制。我们认为这 这种方法可能最终导致潜在的治疗策略的发展，以克服负担， 心脏病

Project Summary Various G-protein-coupled receptors elicit distinct functional responses within a cell, even though they use the common diffusible second messenger cAMP. For instance, while stimulation of either β-adrenergic receptors or E-type prostaglandin receptors leads to cAMP production, only β-adrenergic receptors regulate cardiac myocyte contractility. The ability of a cell to distinguish between cAMP produced in the same cell can only be explained if engagement of different receptors generates distinct receptor-specific pools of cAMP. However, the underlying mechanisms responsible for creating compartmentalized cAMP are not completely understood. Compartmentalized cAMP signaling regulates cardiac contractility and thus is essential for normal functioning of the heart. Consistent with this, dysregulation of cAMP compartmentalization has been linked to several cardiovascular diseases, including cardiac arrhythmias, hypertrophy, and heart failure. Most previous studies have focused on activities of phosphodiesterases, the enzymes that breakdown cAMP, to explain cAMP compartmentation. However, several mathematical studies have predicted that PDE activity alone is not sufficient. These studies have suggested that the mobility of cAMP must be slower than free diffusion to prevent cAMP from reaching non-specific target proteins. We have recently demonstrated that the intracellular mobility of cAMP is markedly hampered by buffering mediated by mitochondria-associated protein kinase A. Now, a new computational study has predicted that, in addition to slow diffusion of cAMP, anatomically restricted spaces within a cell are key to hindering cAMP movement. In cardiac myocytes, mitochondria occupy 30% of the cell volume and are associated with constrained spaces through interactions with the sarcoplasmic reticulum and cytoskeletal proteins. The overall aim of this proposal is to explore the concept that the tight spaces associated with mitochondria regulate cAMP compartmentation. The tethering of mitochondria to the sarcoplasmic reticulum by the proteins, mitofusin-2 (MFN2), glucose-regulated protein 75 (GRP75), and phosphofurin acidic cluster sorting protein 2 (PACS2), creates tight spaces between these organelles. In the FIRST AIM of this study, we will test the hypothesis that the anatomically restricted spaces between mitochondria and the sarcoplasmic reticulum hinder cAMP movement and contribute to cAMP compartmentation. In cardiac myocytes, mitochondrial arrangement is regulated by microtubules and muscle LIM protein (MLP). Disruption of microtubules or MLP causes disorganization of mitochondria and alters mitochondrial morphology, thereby changing the cytosolic spaces associated with mitochondria. Thus, in the SECOND AIM, we hypothesize that cAMP compartmentation is hampered following mitochondrial derangement in microtubule-disrupted cells. To test these hypotheses, we adopt multipronged and complementary approaches to study cAMP compartmentation. Using a variety of advanced techniques, we will measure cAMP mobility, changes in cAMP levels within specific intracellular locations, changes in Ca2+ channel currents and intracellular Ca2+ transients, and test changes in functional responses, such as cell shortening, following stimulation of individual receptors. The goal of this proposal is to elucidate the fundamental mechanisms responsible for facilitating cAMP compartmentation. We believe that this approach may ultimately lead to the development of potential therapeutic strategies to overcome the burden of cardiac diseases.