Hirt Marc N, Boeddinghaus Jasper, Mitchell Alice, Schaaf Sebastian, Börnchen Christian, Müller Christian, Schulz Herbert, Hubner Norbert, Stenzig Justus, Stoehr Andrea, Neuber Christiane, Eder Alexandra, Luther Pradeep K, Hansen Arne, Eschenhagen Thomas
Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; DZHK (German Centre for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, Germany.
Faculty of Medicine, National Heart & Lung Institute, Imperial College London, United Kingdom.
J Mol Cell Cardiol. 2014 Sep;74:151-61. doi: 10.1016/j.yjmcc.2014.05.009. Epub 2014 May 19.
Spontaneously beating engineered heart tissue (EHT) represents an advanced in vitro model for drug testing and disease modeling, but cardiomyocytes in EHTs are less mature and generate lower forces than in the adult heart. We devised a novel pacing system integrated in a setup for videooptical recording of EHT contractile function over time and investigated whether sustained electrical field stimulation improved EHT properties. EHTs were generated from neonatal rat heart cells (rEHT, n=96) or human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hEHT, n=19). Pacing with biphasic pulses was initiated on day 4 of culture. REHT continuously paced for 16-18 days at 0.5Hz developed 2.2× higher forces than nonstimulated rEHT. This was reflected by higher cardiomyocyte density in the center of EHTs, increased connexin-43 abundance as investigated by two-photon microscopy and remarkably improved sarcomere ultrastructure including regular M-bands. Further signs of tissue maturation include a rightward shift (to more physiological values) of the Ca(2+)-response curve, increased force response to isoprenaline and decreased spontaneous beating activity. Human EHTs stimulated at 2Hz in the first week and 1.5Hz thereafter developed 1.5× higher forces than nonstimulated hEHT on day 14, an ameliorated muscular network of longitudinally oriented cardiomyocytes and a higher cytoplasm-to-nucleus ratio. Taken together, continuous pacing improved structural and functional properties of rEHTs and hEHTs to an unprecedented level. Electrical stimulation appears to be an important step toward the generation of fully mature EHT.
自发性搏动的工程心脏组织(EHT)是用于药物测试和疾病建模的先进体外模型,但EHT中的心肌细胞比成年心脏中的心肌细胞成熟度更低,产生的力量也更小。我们设计了一种集成在用于随时间对EHT收缩功能进行视频光学记录的装置中的新型起搏系统,并研究了持续电场刺激是否能改善EHT的特性。EHT由新生大鼠心脏细胞(rEHT,n = 96)或人诱导多能干细胞(hiPSC)衍生的心肌细胞(hEHT,n = 19)生成。在培养第4天开始用双相脉冲进行起搏。连续16 - 18天以0.5Hz起搏的rEHT产生的力量比未刺激的rEHT高2.2倍。这体现在EHT中心更高的心肌细胞密度、通过双光子显微镜研究发现的连接蛋白43丰度增加以及包括规则M带在内的肌节超微结构显著改善。组织成熟的进一步迹象包括Ca(2 +)反应曲线向右移动(至更生理的值)、对异丙肾上腺素的力量反应增加以及自发搏动活动减少。在第一周以2Hz刺激、此后以1.5Hz刺激的人EHT在第14天产生的力量比未刺激的hEHT高1.5倍,纵向排列的心肌细胞的肌肉网络得到改善,细胞质与细胞核的比例更高。综上所述,连续起搏将rEHT和hEHT的结构和功能特性提升到了前所未有的水平。电刺激似乎是朝着生成完全成熟的EHT迈出的重要一步。