Compartmentalized cGMP signalling in cardiomyocyte hypertrophy and heart failure

心肌细胞肥大和心力衰竭中的区室化 cGMP 信号传导

• 批准号: 234439173
• 负责人: Professor Dr. Viacheslav Nikolaev
• 金额: --
• 依托单位: Institut für Experimentelle Herz-Kreislaufforschung
• 依托单位国家: 德国
• 项目类别: Research Units
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/234439173?language=en
• 关键词: Compartmentalized; cGMP; signalling; cardiomyocyte; hypertrophy

The ubiquitous second messenger cGMP regulates multiple cellular processes including smooth and heart muscle contractility, pathological cardiac growth (hypertrophy) and remodelling. In our previous work, we uncovered that hypertrophy is associated with relocation of the cGMP-regulated phosphodiesterases (PDEs) 2 and 3 between beta-adrenoceptor (beta-AR)-associated submembrane microdomains. This newly identified molecular mechanism allows augmentation of catecholamine-stimulated cardiac contractility by atrial natriuretic peptide (ANP) through its receptor GC-A located in T-tubules of cardiomyocytes, where we could also localise functional beta3-ARs. This microdomain might bring together distinct guanylyl cyclases (GC-A/B and NO-GC) to generate an important local pool of cGMP which regulates contraction and pathological remodelling. cGMP microdomain remodelling is a stage-dependent process which involves dynamic changes of cGMP/cAMP cross-talk due to PDE and beta-AR redistribution. Based on our previous work and recently established mouse lines, we will now continue to analyse changes of submembrane cGMP signalling in hypertrophy and heart failure. Using Förster resonance energy transfer and scanning ion conductance microscopy in pm-DE5 mouse cells (expressing a cGMP sensor targeted to T-tubules and caveolin-rich membrane microdomains), we will study whether hypertrophy leads to changes of PDE2-dependent GC-A/cGMP and PDE3-dependent GC-B/cGMP microdomain regulation in the T-tubules vs cell crests. Potentially, GC-A desensitisation and redistribution at the later transition to chronic disease will be analysed together with other FOR 2060 projects. Secondly, we will use pm-DE5 and pm-Epac1-camps animals bred with beta3-AR transgenic mice to study functional interactions of beta3-AR and GC-A receptors in the T-tubular compartment and the regulation of cGMP/cAMP cross-talk by their respective, presumably distinct cGMP pools in the context of disease. Finally, findings on cGMP dynamics obtained in single isolated cardiomyocytes will be further verified using newly established FRET imaging in intact Langendorff perfused hearts with transgenic sensor expression. In this experimental setting, cell type-specific effects in cardiomyocytes, cardiac fibroblasts and potentially also in endothelial cells can be studied and the role of cell-cell communication in the context of pharmacological treatments (such as by sildenafil or drugs acting via NO-GC) can be evaluated. The long-term goal of this project is to identify new druggable targets at the level of subcellular cGMP microdomains, which can be used to prevent cardiac hypertrophy and heart failure progression.

普遍存在的第二信使cGMP调节多种细胞过程，包括平滑肌和心肌收缩性、病理性心脏生长（肥大）和重塑。在我们以前的工作中，我们发现肥大与cGMP调节的磷酸二酯酶（PDE）2和3在β-肾上腺素受体（β-AR）相关的亚膜微区之间的重新定位有关。这种新发现的分子机制允许心房利钠肽（ANP）通过其位于心肌细胞T小管中的受体GC-A增强儿茶酚胺刺激的心脏收缩力，在那里我们也可以定位功能性β 3-AR。该微结构域可能将不同的鸟苷酸环化酶（GC-A/B和NO-GC）聚集在一起，以产生调节收缩和病理重塑的重要局部cGMP库。cGMP微区重构是一个阶段依赖性过程，其涉及由于PDE和β-AR再分布引起的cGMP/cAMP串扰的动态变化。基于我们以前的工作和最近建立的小鼠品系，我们现在将继续分析肥大和心力衰竭中膜下cGMP信号的变化。使用Förster共振能量转移和扫描离子电导显微镜在pm-DE 5小鼠细胞（表达一种靶向T-小管和小窝蛋白丰富的膜微区的cGMP传感器），我们将研究肥大是否导致T-小管与细胞嵴中PDE 2依赖性GC-A/cGMP和PDE 3依赖性GC-B/cGMP微区调节的变化。潜在的，GC-A脱敏和再分布在后期过渡到慢性病将与其他2060项目一起分析。其次，我们将使用pm-DE 5和pm-Epac 1-camp动物与β 3-AR转基因小鼠一起饲养，以研究β 3-AR和GC-A受体在T管室中的功能相互作用以及在疾病背景下通过其各自的、可能不同的cGMP库对cGMP/cAMP串扰的调节。最后，在单个分离的心肌细胞中获得的cGMP动力学的研究结果将进一步验证使用新建立的FRET成像在完整的Langendorff灌注心脏与转基因传感器表达。在这种实验环境中，可以研究心肌细胞、心脏成纤维细胞以及可能的内皮细胞中的细胞类型特异性效应，并且可以评估细胞间通讯在药理学治疗（例如通过西地那非或通过NO-GC作用的药物）背景下的作用。该项目的长期目标是在亚细胞cGMP微结构域水平上确定新的可药用靶点，可用于预防心脏肥大和心力衰竭进展。