Cyclic AMP imaging in adult cardiac myocytes reveals far-reaching β1-adrenergic but locally confined β2-adrenergic receptor-mediated signaling
Cyclic AMP imaging in adult cardiac myocytes reveals far-reaching β1-adrenergic but locally confined β2-adrenergic receptor-mediated signaling
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DOI：
10.1161/01.res.0000250046.69918.d5
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发表时间：
2006-11-10
影响因子：
20.1
通讯作者：
Engelhardt, Stefan
中科院分区：
文献类型：
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作者：
Nikolaev, Viacheslav O.;Buenemann, Moritz;Engelhardt, Stefan
已知β（1）和β（2）-肾上腺素能受体（β AR）差异地调节心肌细胞收缩和生长。我们测试的假设，这些差异是由于第二信使cAMP的空间区室。使用荧光共振能量转移（FRET）为基础的方法，我们直接监测的空间和时间分布的cAMP在成年心肌细胞。我们开发了一种新的cAMP-FRET传感器（称为HCN 2-cams）的基础上的一个单一的cAMP结合结构域的超极化激活的环核苷酸门控钾通道2（HCN 2）。它的胞质分布，高动态范围和灵敏度使HCN 2-营地特别适合监测心肌细胞cAMP的亚细胞定位。我们产生了HCN 2-camp转基因小鼠，并对新鲜分离的心肌细胞进行单细胞FRET成像。异丙肾上腺素全细胞灌注显示cAMP中度升高。各种磷酸二酯酶（PDE）抑制剂的应用显示通过PDE 4> PDE 2> PDE 3严格控制cAMP。β（1）AR介导的cAMP信号完全依赖于PDE 4活性，而β（2）AR介导的cAMP受多种PDE亚型的控制。β（1）AR亚型特异性刺激产生的cAMP反应比选择性β（2）亚型刺激高约2倍，甚至在用非选择性PDE抑制剂3-异丁基-1-甲基黄嘌呤（IBMX）治疗时也是如此（Delta FRET，17.3 +/- 1.3% [β（1）AR] vs 8.8 +/- 0.4% [β（2）AR]）。用百日咳毒素处理BMPG（i）不影响cAMP的产生。局部β（1）AR刺激产生的cAMP梯度在整个细胞中传播，而局部β（2）AR刺激没有引起明显的cAMP扩散。我们的数据显示，在成年心肌细胞中，β（1）AR诱导的cAMP信号影响深远，而β（2）AR诱导的cAMP仍然局限于局部。
beta(1) and beta(2)-adrenergic receptors (beta ARs) are known to differentially regulate cardiomyocyte contraction and growth. We tested the hypothesis that these differences are attributable to spatial compartmentation of the second messenger cAMP. Using a fluorescent resonance energy transfer (FRET)-based approach, we directly monitored the spatial and temporal distribution of cAMP in adult cardiomyocytes. We developed a new cAMP-FRET sensor (termed HCN2-camps) based on a single cAMP binding domain of the hyperpolarization activated cyclic nucleotide-gated potassium channel 2 (HCN2). Its cytosolic distribution, high dynamic range, and sensitivity make HCN2-camps particularly well suited to monitor subcellular localization of cardiomyocyte cAMP. We generated HCN2-camps transgenic mice and performed single-cell FRET imaging on freshly isolated cardiornyocytes. Whole-cell superfusion with isoproterenol showed a moderate elevation of cAMP. Application of various phosphodiesterase (PDE) inhibitors revealed stringent control of cAMP through PDE4 > PDE2 > PDE3. The beta(1)AR-mediated cAMP signals were entirely dependent on PDE4 activity, whereas beta(2)AR-mediated cAMP was under control of multiple PDE isoforms. beta(1)AR subtype-specific stimulation yielded approximate to 2-fold greater cAMP responses compared with selective beta(2)-subtype stimulation, even on treatment with the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) (Delta FRET, 17.3 +/- 1.3% [beta(1)AR] versus 8.8 +/- 0.4% [beta(2)AR]). Treatment with pertussis toxin to inactivate G(i) did not affect cAMP production. Localized beta(1)AR stimulation generated a cAMP gradient propagating throughout the cell, whereas local beta(2)AR stimulation did not elicit marked cAMP diffusion. Our data reveal that in adult cardiac myocytes, beta(1)ARs induce far-reaching cAMP signals, whereas beta(2)AR-induced cAMP remains locally confined.