Interactions of Calcium Fluctuations during Cardiomyocyte Contraction with Real-Time cAMP Dynamics Detected by FRET
Interactions of Calcium Fluctuations during Cardiomyocyte Contraction with Real-Time cAMP Dynamics Detected by FRET
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DOI：
10.1371/journal.pone.0167974
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发表时间：
2016-12-08
期刊：
影响因子：
3.7
通讯作者：
Nikolaev, Viacheslav O.
中科院分区：
文献类型：
--
作者：
Sprenger, Julia U.;Bork, Nadja I.;Nikolaev, Viacheslav O.
钙离子（Ca ~（2+））和环磷酸腺苷（cAMP）在心脏兴奋-收缩-偶联中起关键作用。已知这两种第二信使相互作用，例如通过磷酸二酯酶1（PDE 1）和腺苷酸环化酶5/6（AC 5/6）活性的Ca 2+依赖性调节，这被认为特别是在不同的亚细胞微区的局部水平上发生。目前，许多研究分析了缺乏电刺激的静息心肌细胞中的整体和局部cAMP信号及其调节。例如，福斯特共振能量转移（FRET）显微术是用于在静息心肌细胞中进行的真实的时间cAMP动力学可视化以避免潜在的收缩相关运动伪影的流行方法。然而，尚不清楚这些数据是否与收缩期间在更生理相关条件下的细胞行为相当。在这里，我们直接比较了cAMP-FRET反应AC刺激和PDE抑制静息与起搏的成年小鼠心室心肌细胞的胞质和肌膜下的微域。有趣的是，在β-肾上腺素能（异丙肾上腺素）刺激后未检测到cAMP动力学的显著差异，表明快速变化的收缩Ca 2+浓度对与AC激活相关的胞质cAMP水平的影响较低。然而，钙依赖性的PDE 1，但不是钙不敏感的PDE 4，对毛喉素刺激后cAMP水平的调节的贡献显着增加。这种增加可以通过用Ca 2+升高剂预处理静息细胞来模拟。钙离子成像显示毛喉素中钙瞬变的幅度明显高于异丙肾上腺素刺激的细胞，表明毛喉素刺激可能导致更强的PDE 1激活。总之，心肌细胞收缩过程中细胞内Ca 2+的变化与cAMP水平动态相互作用，特别是在强AC刺激后。在β-肾上腺素能刺激下，使用静息细胞进行基于FRET的cAMP测量是合理的，而可靠的PDE 1效应分析可能需要电场刺激。
Calcium (Ca2+) and 3',5'-cyclic adenosine monophosphate (cAMP) play a critical role for cardiac excitation-contraction-coupling. Both second messengers are known to interact with each other, for example via Ca2+-dependent modulation of phosphodiesterase 1 (PDE1) and adenylyl cyclase 5/6 (AC 5/6) activities, which is supposed to occur especially at the local level in distinct subcellular microdomains. Currently, many studies analyze global and local cAMP signaling and its regulation in resting cardiomyocytes devoid of electrical stimulation. For example, Forster resonance energy transfer (FRET) microscopy is a popular approach for visualization of real time cAMP dynamics performed in resting cardiomyocytes to avoid potential contractility-related movement artifacts. However, it is unknown whether such data are comparable with the cell behavior under more physiologically relevant conditions during contraction. Here, we directly compare the cAMP-FRET responses to AC stimulation and PDE inhibition in resting vs. paced adult mouse ventricular cardiomyocytes for both cytosolic and subsarcolemmal microdomains. Interestingly, no significant differences in cAMP dynamics could be detected after beta-adrenergic (isoproterenol) stimulation, suggesting low impact of rapidly changing contractile Ca2+ concentrations on cytosolic cAMP levels associated with AC activation. However, the contribution of the calcium-dependent PDE1, but not of the Ca2+-insensitive PDE4, to the regulation of cAMP levels after forskolin stimulation was significantly increased. This increase could be mimicked by pretreatment of resting cells with Ca2+ elevating agents. Ca2+ imaging demonstrated significantly higher amplitudes of Ca2+ transients in forskolin than in isoproterenol stimulated cells, suggesting that forskolin stimulation might lead to stronger activation of PDE1. In conclusion, changes in intracellular Ca2+ during cardiomyocyte contraction dynamically interact with cAMP levels, especially after strong AC stimulation. The use of resting cells for FRET-based measurements of cAMP can be justified under beta-adrenergic stimulation, while the reliable analysis of PDE1 effects may require electric field stimulation.