Pijnappels Daniël A, Schalij Martin J, van Tuyn John, Ypey Dirk L, de Vries Antoine A F, van der Wall Ernst E, van der Laarse Arnoud, Atsma Douwe E
Department of Cardiology, Leiden University Medical Center, Leiden, The Netherlands.
Cardiovasc Res. 2006 Nov 1;72(2):282-91. doi: 10.1016/j.cardiores.2006.07.016. Epub 2006 Jul 29.
The purpose of the study was to investigate the development of electrical transmission across human adult bone marrow-derived mesenchymal stem cells (hMSCs) during long-term co-incubation with cardiomyocytes (CMCs).
Neonatal rat CMCs were cultured in multi-electrode array dishes. A conduction block was induced by creating a central acellular channel, yielding two asynchronously beating CMC fields. Enhanced green fluorescent protein (eGFP)-labeled hMSCs from ischemic heart disease patients (n=8), eGFP-labeled hMSCs having RNA interference-mediated connexin43 (Cx43) knockdown (n=6), 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (Dil)-labeled CMCs (n=6), or no cells (n=9) were seeded in the channel. Assessment of conduction velocity (CV), Cx expression and localization, gap junctional coupling, and intracellular electrical recordings were performed for up to 14 days.
Resynchronization of the two CMC fields occurred within 24 h after seeding of hMSCs. CV across hMSCs increased from 1.4+/-0.4 cm/s at day 7 to 3.5+/-0.1 cm/s (p<0.05) at day 14. CV across seeded CMCs was 16.8+/-0.2 cm/s throughout this period. No resynchronization occurred in the absence of seeded cells. Knockdown of Cx43 in hMSCs abolished conduction across the channel completely. Time-dependent increase of CV across hMSCs was associated with increased Cx43 mRNA and protein expression resulting in increased gap junctional coupling. Intracellular recordings in coupled hMSCs showed increased conducted action potential (AP) amplitude, lower resting membrane potential, and decreased duration of conducted AP (p<0.05, day 14 versus day 1).
CV across hMSCs increases progressively after 7 days of co-incubation with CMCs, most likely via improved electrotonic interaction. This is associated with increased Cx43 expression, increased functional gap junctional coupling, and enhanced intercellular electrical coupling between hMSCs and CMCs.
本研究旨在探讨人成年骨髓间充质干细胞（hMSCs）与心肌细胞（CMCs）长期共培养过程中电传导的发展情况。
将新生大鼠CMCs培养在多电极阵列培养皿中。通过创建中央无细胞通道诱导传导阻滞，产生两个异步跳动的CMCs区域。将来自缺血性心脏病患者的增强型绿色荧光蛋白（eGFP）标记的hMSCs（n = 8）、具有RNA干扰介导的连接蛋白43（Cx43）敲低的eGFP标记的hMSCs（n = 6）、1,1'-二辛基-3,3,3',3'-四甲基吲哚碳菁（Dil）标记的CMCs（n = 6）或无细胞（n = 9）接种到通道中。对传导速度（CV）、Cx表达和定位、缝隙连接耦合以及细胞内电记录进行长达14天的评估。
接种hMSCs后24小时内，两个CMCs区域发生了再同步。hMSCs的CV从第7天的1.4±0.4 cm/s增加到第14天的3.5±0.1 cm/s（p<0.05）。在此期间，接种的CMCs的CV为16.8±0.2 cm/s。在没有接种细胞的情况下未发生再同步。hMSCs中Cx43的敲低完全消除了通道的传导。hMSCs的CV随时间增加与Cx43 mRNA和蛋白质表达增加相关，导致缝隙连接耦合增加。耦合hMSCs中的细胞内记录显示传导动作电位（AP）幅度增加、静息膜电位降低以及传导AP的持续时间缩短（第14天与第1天相比，p<0.05）。
hMSCs与CMCs共培养7天后，其CV逐渐增加，最可能是通过改善电紧张相互作用。这与Cx43表达增加、功能性缝隙连接耦合增加以及hMSCs与CMCs之间的细胞间电耦合增强有关。
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