From Emmy Noether Group of the DFG, Department of Cardiology and Pneumology, European Heart Research Institute Göttingen, Georg August University Medical Center, University of Göttingen, Göttingen, Germany (K.R.G., J.U.S., R.K.P., J.H.S., S.E.L., V.O.N.); Institute of Physiology, University of Würzburg, Würzburg, Germany (M.K.); Department of Cardiovascular Sciences, National Heart and Lung Institute, Imperial College, London, United Kingdom (J.G.); and Institut de Recherche Experimentale et Clinique (IREC), Pole of Pharmacology and Therapeutics (FATH), University Catholique de Louvain, and Department of Medicine, Cliniques Universitaires Saint-Luc, Brussels, Belgium (J.-L.B.).
Circ Res. 2014 Apr 11;114(8):1235-45. doi: 10.1161/CIRCRESAHA.114.302437. Epub 2014 Mar 5.
3',5'-Cyclic guanosine monophosphate (cGMP) is an important second messenger that regulates cardiac contractility and protects the heart from hypertrophy. However, because of the lack of real-time imaging techniques, specific subcellular mechanisms and spatiotemporal dynamics of cGMP in adult cardiomyocytes are not well understood.
Our aim was to generate and characterize a novel cGMP sensor model to measure cGMP with nanomolar sensitivity in adult cardiomyocytes.
We generated transgenic mice with cardiomyocyte-specific expression of the highly sensitive cytosolic Förster resonance energy transfer-based cGMP biosensor red cGES-DE5 and performed the first Förster resonance energy transfer measurements of cGMP in intact adult mouse ventricular myocytes. We found very low (≈10 nmol/L) basal cytosolic cGMP levels, which can be markedly increased by natriuretic peptides (C-type natriuretic peptide >> atrial natriuretic peptide) and, to a much smaller extent, by the direct stimulation of soluble guanylyl cyclase. Constitutive activity of this cyclase contributes to basal cGMP production, which is balanced by the activity of clinically established phosphodiesterase (PDE) families. The PDE3 inhibitor, cilostamide, showed especially strong cGMP responses. In a mild model of cardiac hypertrophy after transverse aortic constriction, PDE3 effects were not affected, whereas the contribution of PDE5 was increased. In addition, after natriuretic peptide stimulation, PDE3 was also involved in cGMP/cAMP crosstalk.
The new sensor model allows visualization of real-time cGMP dynamics and pharmacology in intact adult cardiomyocytes. Förster resonance energy transfer imaging suggests the importance of well-established and potentially novel PDE-dependent mechanisms that regulate cGMP under physiological and pathophysiological conditions.
3'，5'-环鸟苷单磷酸（cGMP）是一种重要的第二信使，可调节心肌收缩力并保护心脏免受肥大。然而，由于缺乏实时成像技术，因此，成年心肌细胞中 cGMP 的特定亚细胞机制和时空动力学尚不清楚。
本研究旨在生成并表征一种新型 cGMP 传感器模型，以实现对成年心肌细胞中 cGMP 的纳摩尔灵敏度进行测量。
我们生成了具有心肌细胞特异性表达的高灵敏度细胞溶质Förster 共振能量转移（FRET）基 cGMP 生物传感器 red cGES-DE5 的转基因小鼠，并对成年小鼠心室肌细胞中 cGMP 的首次 FRET 测量进行了研究。我们发现基础细胞溶质 cGMP 水平非常低（≈10 nmol/L），可被利钠肽（C 型利钠肽＞心房利钠肽）显著增加，并且可溶性鸟苷酸环化酶的直接刺激作用要小得多。该环化酶的组成性活性有助于基础 cGMP 的产生，而这一过程受到临床中已确立的磷酸二酯酶（PDE）家族的活性的平衡。PDE3 抑制剂西洛他唑显示出特别强的 cGMP 反应。在横主动脉缩窄后的轻度心脏肥大模型中，PDE3 的作用不受影响，而 PDE5 的作用增加。此外，在利钠肽刺激后，PDE3 也参与了 cGMP/cAMP 串扰。
新型传感器模型可用于可视化成年心肌细胞中实时的 cGMP 动力学和药理学。FRET 成像表明，在生理和病理生理条件下，调节 cGMP 的既定和潜在新型 PDE 依赖性机制很重要。
