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 常见的可扩散的第二信使营地。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 伸缩性。细胞区分同一细胞产生的cAMP的能力只有在以下情况下才能解释 不同受体的结合会产生不同的受体特异性cAMP池。然而，潜在的 造成分隔化营地的机制还不完全清楚。 分区化的cAMP信号调节心肌收缩能力，因此对心脏的正常功能是必不可少的 心脏。与此一致的是，营地划分的失调与几个 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 都专注于磷酸二酯酶的活性，这种酶能分解cAMP，以解释cAMP 车厢分隔。然而，一些数学研究预测，光是PDE活动并不是 足够了。这些研究表明，cAMP的流动性必须慢于自由扩散，以防止 CAMP不能与非特异性靶蛋白结合。我们最近证明了细胞内的流动性 CAMP的活性明显受阻于线粒体相关蛋白激酶A介导的缓冲。 计算研究预测，除了cAMP的缓慢扩散外，解剖受限的空间 在一个细胞内是阻碍营地移动的关键。在心肌细胞中，线粒体占细胞的30%。 体积，通过与肌浆网的相互作用与受限的空间相联系 细胞骨架蛋白。这项提议的总体目标是探索这样一个概念，即狭小的空间与 通过线粒体调节cAMP的划分。线粒体与肌浆网的连接 由丝裂原蛋白-2(Mfn2)、葡萄糖调节蛋白75(GRP75)和磷酸呋喃酸性簇 分类蛋白2(PACS2)，在这些细胞器之间创造了紧密的空间。在这项研究的第一个目的中，我们 将检验这样一种假设，即线粒体和肌浆之间的解剖受限空间 网状结构阻碍了cAMP的运动，并有助于cAMP的区隔。在心肌细胞中，线粒体 排列受微管和肌肉LIM蛋白(MLP)的调节。微管断裂或MLP 导致线粒体的解体和改变线粒体的形态，从而改变胞浆 与线粒体相关的间隙。因此，在第二个目标中，我们假设营地划分 在微管破坏的细胞中线粒体排列紊乱后受到阻碍。为了检验这些假设，我们 采取多管齐下和互补的方法来研究营地划分。使用各种不同的 先进的技术，我们将测量cAMP迁移率，特定细胞内cAMP水平的变化 定位、钙通道电流和细胞内钙瞬变的变化，以及测试功能的变化 刺激后的反应，如细胞缩短。这项提议的目标是 阐明促进cAMP区划的基本机制。我们相信这一点 方法可能最终导致开发潜在的治疗策略，以克服 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.