Wells Centre for Pediatric Research, Indiana University School of Medicine, 1044 West Walnut Street, Indianapolis, IN 46202, USA.
Institute of Biomedical Engineering, National Chiao Tung University, Hsinchu, Taiwan.
Cardiovasc Res. 2018 Mar 1;114(3):389-400. doi: 10.1093/cvr/cvx163.
Recent studies have demonstrated electrotonic coupling between scar tissue and the surrounding myocardium in cryoinjured hearts. However, the electrical dynamics occurring at the myocyte-nonmyocyte interface in the fibrotic heart remain undefined. Here, we sought to develop an assay to interrogate the nonmyocyte cell type contributing to heterocellular coupling and to characterize, on a cellular scale, its voltage response in the infarct border zone of living hearts.
We used two-photon laser scanning microscopy in conjunction with a voltage-sensitive dye to record transmembrane voltage changes simultaneously from cardiomyocytes and adjoined nonmyocytes in Langendorff-perfused mouse hearts with healing myocardial infarction. Transgenic mice with cardiomyocyte-restricted expression of a green fluorescent reporter protein underwent permanent coronary artery ligation and their hearts were subjected to voltage imaging 7-10 days later. Reporter-negative cells, i.e. nonmyocytes, in the infarct border zone exhibited depolarizing transients at a 1:1 coupling ratio with action potentials recorded simultaneously from adjacent, reporter-positive ventricular myocytes. The electrotonic responses in the nonmyocytes exhibited slower rates of de- and repolarization compared to the action potential waveform of juxtaposed myocytes. Voltage imaging in infarcted hearts expressing a fluorescent reporter specifically in myofibroblasts revealed that the latter were electrically coupled to border zone myocytes. Their voltage transient properties were indistinguishable from those of nonmyocytes in hearts with cardiomyocyte-restricted reporter expression. The density of connexin43 expression at myofibroblast-cardiomyocyte junctions was ∼5% of that in the intercalated disc regions of paired ventricular myocytes in the remote, uninjured myocardium, whereas the ratio of connexin45 to connexin43 expression levels at heterocellular contacts was ∼1%.
Myofibroblasts contribute to the population of electrically coupled nonmyocytes in the infarct border zone. The slower kinetics of myofibroblast voltage responses may reflect low electrical conductivity across heterocellular junctions, in accordance with the paucity of connexin expression at myofibroblast-cardiomyocyte contacts.
最近的研究表明，在冷冻损伤的心脏中，疤痕组织与周围心肌之间存在电耦合。然而，纤维化心脏中心肌细胞-非心肌细胞界面的电动力学仍未定义。在这里，我们试图开发一种检测方法来研究对异细胞耦联有贡献的非心肌细胞类型，并在活心脏的梗死边界区从细胞尺度上对其电压反应进行特征描述。
我们使用双光子激光扫描显微镜结合电压敏感染料，同时记录 Langendorff 灌注的具有愈合性心肌梗死的小鼠心脏中心肌细胞和毗邻非心肌细胞的跨膜电压变化。具有心肌细胞特异性绿色荧光报告蛋白表达的转基因小鼠接受永久性冠状动脉结扎，其心脏在 7-10 天后进行电压成像。在梗死边界区，报告蛋白阴性细胞（即非心肌细胞）与同时记录的相邻、报告蛋白阳性心室肌细胞的动作电位以 1:1 的耦联比呈现去极化瞬变。与毗邻心肌细胞的动作电位波形相比，非心肌细胞的电反应表现出较慢的去极化和复极化速率。在表达荧光报告蛋白的梗死心脏中进行电压成像，该报告蛋白特异性表达于肌成纤维细胞中，表明后者与边界区心肌细胞电耦联。它们的电压瞬变特性与在具有心肌细胞特异性报告蛋白表达的心脏中非心肌细胞的特性无法区分。在远程未受伤心肌中，肌成纤维细胞-心肌细胞连接的连接蛋白 43 表达密度约为配对心室肌细胞闰盘区域的 5%，而异细胞接触处连接蛋白 45 与连接蛋白 43 的表达水平比值约为 1%。
肌成纤维细胞有助于梗死边界区电耦联的非心肌细胞群体。肌成纤维细胞电压反应的较慢动力学可能反映了异细胞连接的电导率较低，与肌成纤维细胞-心肌细胞接触处连接蛋白表达不足一致。
