Transgenic Mice for Real-Time Visualization of cGMP in Intact Adult Cardiomyocytes
Transgenic Mice for Real-Time Visualization of cGMP in Intact Adult Cardiomyocytes
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DOI：
10.1161/circresaha.114.302437
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发表时间：
2014-04-11
影响因子：
20.1
通讯作者：
Nikolaev, Viacheslav O.
中科院分区：
文献类型：
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作者：
Goetz, Konrad R.;Sprenger, Julia U.;Nikolaev, Viacheslav O.
理论基础：3，5-环鸟苷一磷酸(CGMP)是一种重要的第二信使，调节心肌收缩能力，保护心脏免受肥大的影响。然而，由于缺乏实时成像技术，对cGMP在成年心肌细胞中的具体亚细胞机制和时空动力学还没有很好的了解。目的：我们的目的是建立和表征一种新型的cGMP传感器模型，以测量成年心肌细胞cGMP的纳摩尔敏感性。方法和结果：我们建立了心肌细胞特异性表达基于胞浆高灵敏Forster共振能量转移的cGMP生物传感器Red CGES-DE5的转基因小鼠，并首次在完整的成年小鼠心室肌细胞中进行了cGMP的Forster共振能量转移测量。我们发现细胞内cGMP的基础水平很低(约10nmol/L)，可被利钠肽(C型利钠肽和心房利钠肽)显著升高，而直接刺激可溶性鸟苷酸环酶的作用要小得多。这种环化酶的组成活性有助于基础cGMP的产生，而cGMP的产生被临床上建立的磷酸二酯酶(PDE)家族的活性所平衡。PDE3抑制剂西洛斯塔胺表现出特别强的cGMP反应。在一种轻度心肌肥厚模型中，PDE3的作用不受影响，而PDE5的作用增加。此外，在钠尿肽刺激后，PDE3也参与了cGMP/cAMP交叉通路。结论：新的传感器模型可以实时可视化完整成年心肌细胞中cGMP的动力学和药理学。Forster共振能量转移成像表明，在生理和病理生理条件下调节cGMP的已建立的和潜在的新型PDE依赖机制的重要性。
Rationale: 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.Objective: Our aim was to generate and characterize a novel cGMP sensor model to measure cGMP with nanomolar sensitivity in adult cardiomyocytes.Methods and Results: We generated transgenic mice with cardiomyocyte-specific expression of the highly sensitive cytosolic Forster resonance energy transfer-based cGMP biosensor red cGES-DE5 and performed the first Forster resonance energy transfer measurements of cGMP in intact adult mouse ventricular myocytes. We found very low (approximate to 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.Conclusions: The new sensor model allows visualization of real-time cGMP dynamics and pharmacology in intact adult cardiomyocytes. Forster resonance energy transfer imaging suggests the importance of well-established and potentially novel PDE-dependent mechanisms that regulate cGMP under physiological and pathophysiological conditions.