McSpadden Luke C, Kirkton Robert D, Bursac Nenad
Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA.
Am J Physiol Cell Physiol. 2009 Aug;297(2):C339-51. doi: 10.1152/ajpcell.00024.2009. Epub 2009 Jun 3.
Understanding how electrotonic loading of cardiomyocytes by unexcitable cells alters cardiac impulse conduction may be highly relevant to fibrotic heart disease. In this study, we optically mapped electrical propagation in confluent, aligned neonatal rat cardiac monolayers electrotonically loaded with cardiac fibroblasts, control human embryonic kidney (HEK-293) cells, or HEK-293 cells genetically engineered to overexpress the gap junction proteins connexin-43 or connexin-45. Gap junction expression and function were assessed by immunostaining, immunoblotting, and fluorescence recovery after photobleaching and were correlated with the optically mapped propagation of action potentials. We found that neonatal rat ventricular fibroblasts negative for the myofibroblast marker smooth muscle alpha-actin expressed connexin-45 rather than connexin-43 or connexin-40, weakly coupled to cardiomyocytes, and, without significant depolarization of cardiac resting potential, slowed cardiac conduction to 75% of control only at high (>60%) coverage densities, similar to loading effects found from HEK-293 cells expressing similar levels of connexin-45. In contrast, HEK-293 cells with connexin-43 expression similar to that of cardiomyocytes significantly decreased cardiac conduction velocity and maximum capture rate to as low as 22% and 25% of control values, respectively, while increasing cardiac action potential duration to 212% of control and cardiac resting potential from -71.6 +/- 4.9 mV in controls to -65.0 +/- 3.8 mV. For all unexcitable cell types and coverage densities, velocity anisotropy ratio remained unchanged. Despite the induced conduction slowing, none of the loading cell types increased the proportion of spontaneously active monolayers. These results signify connexin isoform and expression level as important contributors to potential electrical interactions between unexcitable cells and myocytes in cardiac tissue.
了解不可兴奋细胞对心肌细胞的电紧张性负荷如何改变心脏冲动传导可能与纤维化心脏病密切相关。在本研究中，我们利用光学映射技术研究了汇合、排列的新生大鼠心脏单层细胞中的电传播情况，这些单层细胞被电紧张性加载了心脏成纤维细胞、对照人胚肾（HEK - 293）细胞，或经基因工程改造过表达缝隙连接蛋白连接蛋白 - 43或连接蛋白 - 45的HEK - 293细胞。通过免疫染色、免疫印迹和光漂白后的荧光恢复来评估缝隙连接的表达和功能，并将其与光学映射的动作电位传播相关联。我们发现，肌成纤维细胞标志物平滑肌α - 肌动蛋白呈阴性的新生大鼠心室成纤维细胞表达连接蛋白 - 45而非连接蛋白 - 43或连接蛋白 - 40，与心肌细胞的耦合较弱，并且在心脏静息电位无明显去极化的情况下，仅在高（>60%）覆盖密度时才将心脏传导减慢至对照的75%，这与表达相似水平连接蛋白 - 45的HEK - 293细胞的加载效应相似。相比之下，连接蛋白 - 43表达水平与心肌细胞相似的HEK - 293细胞显著降低心脏传导速度和最大捕获率，分别低至对照值的22%和25%，同时将心脏动作电位持续时间增加至对照的212%，并将心脏静息电位从对照的 - 71.6±4.9 mV变为 - 65.0±3.8 mV。对于所有不可兴奋细胞类型和覆盖密度，速度各向异性比率保持不变。尽管诱导了传导减慢，但没有一种加载细胞类型增加自发活动单层的比例。这些结果表明，连接蛋白异构体和表达水平是心脏组织中不可兴奋细胞与心肌细胞之间潜在电相互作用的重要因素。
