Calcium handling in human embryonic stem cell-derived cardiomyocytes
Calcium handling in human embryonic stem cell-derived cardiomyocytes
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DOI：
10.1634/stemcells.2007-0591
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发表时间：
2008-08-01
期刊：
影响因子：
5.2
通讯作者：
Gepstein, Lior
中科院分区：
文献类型：
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作者：
Satin, Jonathan;Itzhaki, Ilanit;Gepstein, Lior
本研究的目的是表征发育中的人胚胎干细胞衍生的心肌细胞（hESC-CM）中的钙处理。为此，使用实时聚合酶链反应（PCR），免疫细胞化学，全细胞电压钳，并同时膜片钳/激光扫描共聚焦钙成像和表面膜标记与二-8-氨基萘基乙烯基吡啶。hESC-CM中的免疫染色研究证实了肌浆网（SR）钙释放通道、ryanodine受体-2和肌醇-1，4，5-三磷酸（IP 3）受体的存在。储存钙功能表现为动作电位诱导的钙瞬变。时间到目标图显示，这些动作电位启动的钙瞬变通过细胞内钙释放的传播波穿过细胞的宽度。hESC-CM还表现出定位于表面膜的局部钙事件（“火花”）。咖啡因敏感的细胞内钙储存的存在在三个不同年龄组中应用局灶性、时间有限的咖啡因喷流后得到证实：早期（开始搏动）、中期（搏动后10天[dpb]）和晚期（30-40 dpb）hESC-CM。在离体成熟过程中，钙库负荷逐渐增加。同样，应用兰尼定降低自发钙瞬变的幅度。有趣的是，在使用笼状IP 3光解和拮抗剂应用（2 μ M 2-氨基乙氧基二苯基硼酸盐）的实验中，在hESC-CM中也证明了IP 3可释放的钙库的表达和功能。总之，我们的研究确立了早期hESC-CM中存在功能性SR钙储存，并显示了这些细胞中钙处理的独特模式。这项研究还强调了hESC-CM的功能特性的重要性，无论是发育研究和未来的心肌细胞替代策略的发展。
The objective of the current study was to characterize calcium handling in developing human embryonic stem cell-derived cardiomyocytes (hESC-CMs). To this end, real-time polymerase chain reaction (PCR), immunocytochemistry, whole-cell voltage-clamp, and simultaneous patch-clamp/laser scanning confocal calcium imaging and surface membrane labeling with di-8-aminonaphthylethenylpridinium were used. Immunostaining studies in the hESC-CMs demonstrated the presence of the sarcoplasmic reticulum (SR) calcium release channels, ryanodine receptor-2, and inositol-1,4,5-trisphosphate (IP3) receptors. Store calcium function was manifested as action-potential-induced calcium transients. Time-to-target plots showed that these action-potential-initiated calcium transients traverse the width of the cell via a propagated wave of intracellular store calcium release. The hESC-CMs also exhibited local calcium events ("sparks") that were localized to the surface membrane. The presence of caffeine-sensitive intracellular calcium stores was manifested following application of focal, temporally limited puffs of caffeine in three different age groups: early-stage (with the initiation of beating), intermediate-stage (10 days post-beating [dpb]), and late-stage (30-40 dpb) hESC-CMs. Calcium store load gradually increased during in vitro maturation. Similarly, ryanodine application decreased the amplitude of the spontaneous calcium transients. Interestingly, the expression and function of an IP3-releasable calcium pool was also demonstrated in the hESC-CMs in experiments using caged-IP3 photolysis and antagonist application (2 mu M 2-Aminoethoxydiphenyl borate). In summary, our study establishes the presence of a functional SR calcium store in early-stage hESC-CMs and shows a unique pattern of calcium handling in these cells. This study also stresses the importance of the functional characterization of hESC-CMs both for developmental studies and for the development of future myocardial cell replacement strategies.