Optogenetic activation of Gq signalling modulates pacemaker activity of cardiomyocytes
Optogenetic activation of Gq signalling modulates pacemaker activity of cardiomyocytes
复制标题
DOI：
10.1093/cvr/cvu046
复制
发表时间：
2014-06-01
影响因子：
10.8
通讯作者：
Sasse, Philipp
中科院分区：
文献类型：
--
作者：
Beiert, Thomas;Bruegmann, Tobias;Sasse, Philipp
用G(Q)偶联受体的药物激动剂研究G(Q)信号缺乏时空精确度。本研究的目的是建立一种光敏感的G(Q)偶联受体黑素，作为研究G(Q)刺激对早期发育心肌细胞起搏的时空效应的新工具。在HEK293FT细胞中测试了普遍表达黑素的载体，结果表明光诱导产生肌醇-1，4，5-三磷酸并增加细胞内钙离子浓度。将该质粒稳定地导入小鼠胚胎干细胞，并分化为自发搏动的类胚体(EBS)。EBs内的心肌细胞表达黑素，光照(60 S，308.5 nW/mm(2)，470 nm)的EBS10.2+/-1.7S使搏动频率增加到基线频率的317.1+/-16.3%。S只需5分钟的光照就足以产生最大的频率响应。停止光照后，拍频达到基线频率，其衰减常数为27.1+/-2.5 S，光诱导的拍频加速与光强呈S形关系，半数有效光强为41.7NW/mm(2)。有趣的是，在停止高强度照射后，EBS显示出高比例的不规则收缩。通过照射心肌细胞功能合体中的一小块区域，局部G(Q)激活可导致照光区域内的起搏器活动。在表达黑素的心肌细胞中，光诱导G(Q)激活可增加搏动频率并产生局部起搏器活动。我们认为，在心血管研究中，黑素是一种强大的光遗传学工具，用于研究G(Q)信号的空间和时间方面。
Investigation of G(q) signalling with pharmacological agonists of G(q)-coupled receptors lacks spatio-temporal precision. The aim of this study was to establish melanopsin, a light-sensitive G(q)-coupled receptor, as a new tool for the investigation of spatial and temporal effects of G(q) stimulation on pacemaking in cardiomyocytes at an early developmental stage.A vector for ubiquitous expression of melanopsin was tested in HEK293FT cells, which showed light-induced production of inositol-1,4,5-trisphosphate and elevation of intracellular Ca2+ concentration. Mouse embryonic stem cells were stably transfected with this plasmid and differentiated into spontaneously beating embryoid bodies (EBs). Cardiomyocytes within EBs showed melanopsin expression and illumination (60 s, 308.5 nW/mm(2), 470 nm) of EBs increased beating rate within 10.2 +/- 1.7 s to 317.1 +/- 16.3% of baseline frequency. Illumination as short as 5 s was sufficient for generating the maximal frequency response. After termination of illumination, baseline frequency was reached with a decay constant of 27.1 +/- 2.5 s. The light-induced acceleration of beating frequency showed a sigmoid dependence on light intensity with a half maximal effective light intensity of 41.7 nW/mm(2). Interestingly, EBs showed a high rate of irregular contractions after termination of high-intensity illumination. Local G(q) activation by illumination of a small region in a functional syncytium of cardiomyocytes led to pacemaker activity within the illuminated area.Light-induced G(q) activation in melanopsin-expressing cardiomyocytes increases beating rate and generates local pacemaker activity. We propose that melanopsin is a powerful optogenetic tool for the investigation of spatial and temporal aspects of G(q) signalling in cardiovascular research.