Engineered Heart Slice Model of Arrhythmogenic Cardiomyopathy Using Plakophilin-2 Mutant Myocytes
Engineered Heart Slice Model of Arrhythmogenic Cardiomyopathy Using Plakophilin-2 Mutant Myocytes
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DOI：
10.1089/ten.tea.2018.0272
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发表时间：
2019-05-01
影响因子：
4.1
通讯作者：
Tung, Leslie
中科院分区：
文献类型：
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作者：
Blazeski, Adriana;Lowenthal, Justin;Tung, Leslie
心律失常性心肌病（AC）是一种遗传性疾病，可以在体外利用诱导多能干细胞的患者特异性心肌细胞（CMs）建立模型。了解潜在的疾病机制，特别是在早期隐藏阶段，可能导致新的诊断和治疗策略。然而，需要多细胞合胞模型来了解连接细胞的桥粒的遗传编码突变如何导致异常的电传导和心律失常。在这项研究中，通过将来自一名plakophilin-2 （PKP2）突变的AC患者的人诱导多能干细胞来源的CMs植入到完整的脱细胞心肌切片上，构建了工程心脏切片（EHS），然后与单层培养的年龄匹配的AC CMs进行了比较。培养2周后，EHS发育成融合的多层合胞体，表现出自发的协调跳动，可以在2000至500ms的周期长度范围内进行电节律。作为EHS培养的AC CMs显示出高度排列、致密和有序的肉瘤结构，基因表达分析显示成熟程度增加。此外，在EHS中，CM培养会影响ac相关基因，与单层细胞相比，PPARG显著增加，SCN5A显著减少。在功能上，与单层膜相比，交流EHS具有相似的传导速度、更短的动作电位和更慢、更稳定的自发心率。S1-S2起搏也可诱发再入性心律失常。我们的研究结果表明，EHS微环境增强了AC-CMs在培养中的表型，同时允许对AC-CMs适当排列的合胞体进行功能研究。本文报道的结果证明了使用EHS研究AC的好处，EHS是一种允许合胞培养和结合基质线索的组织结构。遗传性心脏病，如心律失常性心肌病（AC），是心脏性猝死的常见遗传原因，可以使用患者特异性诱导多能干细胞衍生的心肌细胞（CMs）进行建模。然而，由于细胞-细胞和细胞-基质相互作用的重要性，在多细胞合胞体中培养这些细胞并暴露于周围的基质线索，以创建更准确和强大的疾病模型是很重要的。通过在完整的脱细胞基质切片上播种CMs构建工程化心脏切片，可以对CMs的多层合胞体进行分子和功能研究。这项研究揭示了一种改进的疾病培养皿模型的潜力。
Arrhythmogenic cardiomyopathy (AC), a cause of sudden cardiac death among young and otherwise healthy individuals, is a heritable disease that can be modeled in vitro using patient-specific cardiac myocytes (CMs) from induced pluripotent stem cells. An understanding of underlying disease mechanisms, particularly in the early concealed stages, could lead to new diagnosis and treatment strategies. However, multicellular syncytial models are needed to understand how genetically encoded mutations of the desmosomes that interconnect cells lead to aberrant electrical conduction and arrhythmias. In this study, engineered heart slices (EHS) were created by seeding human induced pluripotent stem cell-derived CMs from an AC patient with a plakophilin-2 (PKP2) mutation onto intact slices of decellularized myocardium and then compared to age-matched AC CMs cultured as monolayers. After 2 weeks of culture, EHS developed into a confluent multilayered syncytia that exhibited spontaneous coordinated beating and could be electrically paced at cycle lengths ranging from 2000 to 500ms. AC CMs cultured as EHS displayed highly aligned, dense, and ordered sarcomeric structures, with gene expression analyses revealing increased maturation. In addition, AC-relevant genes were affected by CM culture in EHS, with a substantial increase in PPARG and a decrease in SCN5A compared to monolayers. Functionally, AC EHS exhibited similar conduction velocities, shorter action potentials, and a slower and steadier spontaneous beat rate compared with monolayers. Reentrant arrhythmias could also be induced in AC EHS by S1-S2 pacing. Our findings suggest that the EHS microenvironment enhances the phenotype of AC CMs in culture while allowing for functional studies of an appropriately aligned syncytium of AC-CMs. Results reported here demonstrate the benefits of studying AC using EHS, a tissue construct that allows syncytial culture and the incorporation of matrix cues.Impact Statement Genetic heart diseases such as arrhythmogenic cardiomyopathy (AC), a common genetic cause of sudden cardiac death, can be modeled using patient-specific induced pluripotent stem cell-derived cardiac myocytes (CMs). However, it is important to culture these cells in a multicellular syncytium with exposure to surrounding matrix cues to create more accurate and robust models of the disease due to the importance of cell-cell and cell-matrix interactions. The engineered heart slice, constructed by seeding CMs on intact decellularized matrix slices, allows molecular and functional studies on an aligned multilayered syncytium of CMs. This study reveals the potential for an improved disease-in-a-dish model of AC.