Influence of Electromechanical Activity on Cardiac Differentiation of Mouse Embryonic Stem Cells.
Influence of Electromechanical Activity on Cardiac Differentiation of Mouse Embryonic Stem Cells.
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DOI：
10.1007/s13239-010-0020-8
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发表时间：
2010-09
影响因子：
1.8
通讯作者：
Lipke EA
中科院分区：
文献类型：
--
作者：
Limpitikul W;Christoforou N;Thompson SA;Gearhart JD;Tung L;Lipke EA
在分化过程中，小鼠胚胎干细胞衍生的心肌细胞（mESC-CM）接受自发搏动的机电线索。因此，通过电起搏促进电机械活动或通过药物治疗抑制电机械活动可能会影响细胞功能发育。在分化后期（第16-18天），对融合的mESC-CM单层施加电起搏。或者，通过（a）在第11-18天用CsCl（HCN通道）、曲唑酮（T型Ca 2+通道）或CsCl和曲唑酮阻断离子电流;或（B）在第11-14天应用blebbistatin（兴奋-收缩解偶联剂）来抑制自发收缩。分别在第19天和第18天检查电生理特性和基因表达。光学标测显示起搏与非起搏单层的传导速度（CV）无显著差异，连接蛋白-43、钠钙交换器（NCX）或肌球蛋白重链（MHC）的基因表达也无显著变化。然而，在起搏mESC-CM中，分化批次之间的CV变异性和单个单层内的CV异质性显著较低。或者，虽然四种药物治疗以不同程度抑制收缩（直至完全抑制），但与对照组相比，任何治疗的CV均无显著差异。与对照组相比，曲唑酮治疗显著降低了CV变异性，而CsCl治疗显著降低了CV异质性。未观察到连接蛋白-43、MHC、HCN 1、Cav3.1/3.2的基因表达的明显变化。在晚期分化期间，电起搏而不是抑制自发收缩降低了分化批次之间和单个单层之间CV的固有变异性，这在ESC用于心肌组织修复的应用中可能是有益的。
During differentiation, mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) receive electromechanical cues from spontaneous beating. Therefore, promoting electromechanical activity via electrical pacing or suppressing it by drug treatment might affect the cellular functional development. Electrical pacing was applied to confluent monolayers of mESC-CMs during late-stage differentiation (days 16–18). Alternatively, spontaneous contraction was suppressed by (a) blocking ion currents with CsCl (HCN channel), trazodone (T-type Ca2+ channel), or both CsCl and trazodone on days 11–18; or (b) applying blebbistatin (excitation–contraction uncoupler) on days 11–14. Electrophysiological properties and gene expression were examined on day 19 and 18, respectively. Optical mapping revealed no significant difference in conduction velocity (CV)in paced vs. non-pacedmonolayers, nor were there significant changes in gene expression of connexin-43, Na–Ca exchanger (NCX), or myosin heavy chain (MHC). However, CV variability among differentiation batches and CV heterogeneity within individual monolayers were significantly lower in paced mESC-CMs. Alternatively, while the four drug treatments suppressed contraction with varying degrees (up to complete inhibition), there was no significant difference in CV for any of the treatments compared with controls. Trazodone treatment significantly reduced CV variability as compared to controls, whereas CsCl treatment significantly reduced CV heterogeneity. Distinct changes in gene expression of connexin-43, MHC, HCNl, Cav3.1/3.2 were not observed. Electrical pacing, but not suppression of spontaneous contraction, during late-stage differentiation reduces the intrinsic variability of CV among differentiation batches and across individual monolayers, which can be beneficial in the application of ESCs for myocardial tissue repair.