Compartmentalized cGMP signalling in cardiomyocyte hypertrophy and heart failure

普遍存在的第二信使cGMP调节多种细胞过程，包括平滑和心肌收缩、病理性心脏生长(肥大)和重塑。在我们以前的工作中，我们发现肥厚与cGMP调节的磷酸二酯酶(PDE)2和3在与β-肾上腺素能受体(β-AR)相关的膜下微域之间的重新定位有关。这一新发现的分子机制允许心钠素(ANP)通过其位于心肌细胞T管的受体GC-A增强儿茶酚胺刺激的心肌收缩能力，我们也可以在那里定位功能的β3-AR。这个微域可能结合不同的鸟苷酸环化酶(GC-A/B和NO-GC)，产生一个重要的局部cGMP池，调节收缩和病理重塑。CGMP微区重构是一个阶段依赖的过程，它涉及PDE和β-AR重分布引起的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系，我们现在将继续分析肥厚和心力衰竭时膜下cGMP信号的变化。利用PM-DE5小鼠细胞的Förster共振能量转移和扫描离子电导显微镜(表达针对T小管和富含小窝蛋白的膜微区的cGMP传感器)，我们将研究肥大是否导致T小管与细胞顶中依赖PDE2的GC-A/cGMP和依赖于PDE3的GC-B/cGMP微域调控的变化。潜在地，GC-A脱敏和在向慢性病的后期过渡时的再分配将与2060年的其他项目一起进行分析。其次，我们将使用与Beta3-AR转基因小鼠培育的PM-DE5和PM-Epac1-cAMP动物来研究T管室中β3-AR和GC-A受体的功能相互作用，以及它们各自的cGMP/cAMP串扰在疾病背景下的调节。最后，在单个分离的心肌细胞中获得的cGMP动力学结果将通过新建立的FRET成像在具有转基因传感器表达的完整的朗多夫灌流心脏中得到进一步验证。在这个实验环境中，可以研究心肌细胞、心脏成纤维细胞以及潜在的内皮细胞中特定细胞类型的影响，并可以评估药物治疗(如西地那非或通过NO-GC作用的药物)中细胞-细胞通讯的作用。该项目的长期目标是在亚细胞cGMP微域水平上确定新的可用药靶点，可用于预防心肌肥厚和心力衰竭进展。

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.