Label-free conduction velocity mapping and gap junction assessment of functional iPSC-Cardiomyocyte monolayers
Label-free conduction velocity mapping and gap junction assessment of functional iPSC-Cardiomyocyte monolayers
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DOI：
10.1016/j.bios.2020.112468
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发表时间：
2020-11-01
影响因子：
12.6
通讯作者：
Sun, Yu
中科院分区：
文献类型：
--
作者：
Dou, Wenkun;Zhao, Qili;Sun, Yu
心脏传导是心脏的重要功能。迄今为止，在体外传导速度（CV）的准确测量受到微电极阵列（MEA）的低空间分辨率和低信噪比，或细胞毒性和终点分析的荧光光学成像的阻碍。在这里，我们已经开发了一种新的无标记方法的基础上散焦明场成像量化CV通过分析质心位移和收缩轨迹的每个心肌细胞在一个单层的人干细胞衍生的心肌细胞（iPSC-CM）。我们的数据显示，细胞内钙释放和细胞收缩开始之间的时间延迟在心肌细胞中是高度一致的;然而，细胞达到其最大跳动幅度所需的持续时间差异显着，证明了iPSC-CM中兴奋-收缩偶联的时间延迟在很大程度上是恒定的。进行相同iPSC-CM群体（类似于10（6）个细胞）的标准钙成像以与我们的无标记方法进行比较。结果证实，我们的无标记方法能够实现高度准确的CV标测（17.64 +/- 0.89 cm/s vs. 17.95 +/- 2.29 cm/s，p值>0.1）。此外，我们的方法有效地揭示了各种形状的细胞跳动模式。我们还对具有斑嗜蛋白-2（PKP 2）敲低的疾病特异性iPSC-CM单层进行了无标记CV作图，这有效地量化了它们的低CV值，并进一步验证了PKP 2突变通过破坏心脏传导在致心律失常性右心室心肌病（ARVC）中的致心律失常作用。无标记方法提供了一种无细胞毒性的技术，用于长期测量心肌细胞单层的动态搏动轨迹、搏动传播和传导速度。
Cardiac conduction is an important function of the heart. To date, accurate measurement of conduction velocity (CV) in vitro is hindered by the low spatial resolution and poor signal-to-noise ratio of microelectrode arrays (MEAs), or the cytotoxicity and end-point analysis of fluorescence optical imaging. Here, we have developed a new label-free method based on defocused brightfield imaging to quantify CV by analyzing centroid displacements and contraction trajectories of each cardiomyocyte in a monolayer of human stem cell-derived cardiomyocytes (iPSC-CMs). Our data revealed that the time delay between intracellular calcium release and the initiation of cell contraction is highly consistent across cardiomyocytes; however, the duration a cell takes to reach its maximum beating magnitude varies significantly, proving that the time delay in excitation-contraction coupling is largely constant in iPSC-CMs. Standard calcium imaging of the same iPSC-CM populations (similar to 10(6) cells) was conducted for comparison with our label-free method. The results confirmed that our label-free method was capable of achieving highly accurate CV mapping (17.64 +/- 0.89 cm/s vs. 17.95 +/- 2.29 cm/s, pvalue>0.1). Additionally, our method effectively revealed various shapes in cell beating pattern. We also performed label-free CV mapping on disease-specific iPSC-CM monolayers with plakophilin-2 (PKP2) knockdown, which effectively quantified their low CV values and further validated the arrhythmogenic role of PKP2 mutation in arrhythmogenic right ventricular cardiomyopathy (ARVC) through the disruption of cardiac conduction. The label-free method offers a cytotoxic-free technique for long-term measurement of dynamic beating trajectories, beating propagation and conduction velocities of cardiomyocyte monolayers.