Institute of Physiology I, Medical Faculty, University of Bonn, Nussallee 11, 53115 Bonn, Germany.
Institute for Cardiovascular Physiology, University Medical Center Goettingen, Goettingen, Germany.
Europace. 2020 Oct 1;22(10):1590-1599. doi: 10.1093/europace/euaa128.
Besides providing mechanical stability, fibroblasts in the heart could modulate the electrical properties of cardiomyocytes. Here, we aim to develop a three-dimensional hetero-cellular model to analyse the electric interaction between fibroblasts and human cardiomyocytes in vitro using selective optogenetic de- or hyperpolarization of fibroblasts.
NIH3T3 cell lines expressing the light-sensitive ion channel Channelrhodopsin2 or the light-induced proton pump Archaerhodopsin were generated for optogenetic depolarization or hyperpolarization, respectively, and characterized by patch clamp. Cardiac bodies consisting of 50% fibroblasts and 50% human pluripotent stem cell-derived cardiomyocytes were analysed by video microscopy and membrane potential was measured with sharp electrodes. Myofibroblast activation in cardiac bodies was enhanced by transforming growth factor-β1 (TGF-β1)-stimulation. Connexin-43 expression was analysed by qPCR and fluorescence recovery after photobleaching. Illumination of Channelrhodopsin2 or Archaerhodopsin expressing fibroblasts induced inward currents and depolarization or outward currents and hyperpolarization. Transforming growth factor-β1-stimulation elevated connexin-43 expression and increased cell-cell coupling between fibroblasts as well as increased basal beating frequency and cardiomyocyte resting membrane potential in cardiac bodies. Illumination of cardiac bodies generated with Channelrhodopsin2 fibroblasts accelerated spontaneous beating, especially after TGF-β1-stimulation. Illumination of cardiac bodies prepared with Archaerhodopsin expressing fibroblasts led to hyperpolarization of cardiomyocytes and complete block of spontaneous beating after TGF-β1-stimulation. Effects of light were significantly smaller without TGF-β1-stimulation.
Transforming growth factor-β1-stimulation leads to increased hetero-cellular coupling and optogenetic hyperpolarization of fibroblasts reduces TGF-β1 induced effects on cardiomyocyte spontaneous activity. Optogenetic membrane potential manipulation selectively in fibroblasts in a new hetero-cellular cardiac body model allows direct quantification of fibroblast-cardiomyocyte coupling in vitro.
除了提供机械稳定性外，心脏中的成纤维细胞还可以调节心肌细胞的电特性。在此，我们旨在开发一种三维异细胞模型，以使用选择性光遗传学方法对成纤维细胞去极化或超极化来分析体外成纤维细胞与人类心肌细胞之间的电相互作用。
生成表达光敏感离子通道 Channelrhodopsin2 或光诱导质子泵 Archaerhodopsin 的 NIH3T3 细胞系，分别用于光遗传学去极化或超极化，并通过膜片钳进行表征。通过视频显微镜分析由 50％成纤维细胞和 50％人多能干细胞衍生的心肌细胞组成的心脏体，并使用尖锐电极测量膜电位。心脏体中的成纤维细胞激活通过转化生长因子-β1（TGF-β1）刺激增强。通过 qPCR 和光漂白后荧光恢复分析连接蛋白 43 的表达。光刺激表达 Channelrhodopsin2 或 Archaerhodopsin 的成纤维细胞会引起内向电流和去极化或外向电流和超极化。TGF-β1 刺激可上调连接蛋白 43 的表达，并增加成纤维细胞之间的细胞间偶联，以及增加心脏体中的心肌细胞静息膜电位和基础搏动频率。用光刺激用 Channelrhodopsin2 成纤维细胞制备的心脏体可加速自发性搏动，尤其是在 TGF-β1 刺激后。用光刺激用 Archaerhodopsin 表达成纤维细胞制备的心脏体可导致心肌细胞超极化，并在 TGF-β1 刺激后完全阻断自发性搏动。没有 TGF-β1 刺激时，光的影响要小得多。
TGF-β1 刺激导致异细胞偶联增加，光遗传学超极化成纤维细胞可降低 TGF-β1 对心肌细胞自发性活动的影响。在新的异细胞心脏体模型中，选择性用光遗传学方法对成纤维细胞进行膜电位操作可直接定量体外成纤维细胞-心肌细胞偶联。
