Reverse-engineering the sinoatrial node with induced pacemaker cells

摘要 背景：哺乳动物的心脏自发自主地跳动， （~ 10，000）起搏细胞。尽管我们对个体心脏病患者 起搏细胞自动跳动，缺乏对一些起搏细胞如何能够 驱动整个心脏的跳动。这个问题，被称为“源-汇不匹配”，是一个基本的 由于研究这些起搏器的吞吐量非常低，因此难以研究 细胞这是因为没有可测试的模型的SAN存在，纳入心脏起搏器细胞 和静止心肌细胞。通常情况下，只有少数的天然起搏细胞可以从 天然SAN和分离的细胞不能培养。最近，我的团队证明了 普通心室肌细胞向诱导性起搏细胞（iPCs）的转化 TBX 18的表达。在本提案中，我们寻求通过利用 de novo iPCs。我们假设iPCs的2维和3维结构可以作为体外培养的 原生SAN模型。 方法：我们将研究本机SAN的四个设计原则，i）所需的iPC的最小数量 起搏给定数量的相邻心室肌细胞，ii）非肌细胞群体在 起搏，iii）SAN的形状，以及iv）出口通路的需要。我们的2D模型将包括 模式化的单层，而我们的3D模型使用模式化的心脏球体。使用常规 聚二甲基硅氧烷（PDMS）模板技术，我们将创建一个由一个封闭的iPCs的人口 静止的心室心肌细胞群。主要的读数是i）实时、全细胞Ca 2 + 整个单分子层的瞬态，ii）快速，高分辨率的单分子层的光学映射， 电压敏感染料，和iii）场电位的宏观尺度、多电极阵列测量。我们 初步数据表明IPC-球状体存活至少三周。当一群15-20个 TBX 18球状体被单层心室肌细胞TBX 18包围，但没有GFP（对照）， 球状体能够起搏并驱动邻近的心室肌细胞片层。 成功完成我们的项目可以创建工程SA节点（eSAN）， 概括本机SAN的设计原则。反过来，这项技术提供了一个方便的 在此平台上，可以针对持久性生物起搏器构建其他SAN设计原则。

ABSTRACT Background: The mammalian heart beats spontaneously and autonomously due to few thousand (~10,000) pacemaker cells. Although we have a general understanding of how individual cardiac pacemaker cells beat automatically, there is a lack of understanding in how a few pacemaker cells can drive the beating of the entire heart. This problem, known as a “source-sink mismatch”, is a fundamental concept that has been difficult to study due to it being painfully low-throughput to study these pacemaker cells. This is because no testable model of the SAN exists, incorporating the cardiac pacemaker cells and quiescent cardiomyocytes. Typically, just a handful of native pacemaker cells can be isolated from the native SAN, and the isolated cell cannot be cultured. Recently, my group has demonstrated conversion of ordinary ventricular cardiomyocytes to induced pacemaker cells (iPCs) by singular expression of TBX18. In this proposal, we seek to engineer tissue models of the SAN by exploiting the de novo iPCs. We hypothesize that 2- and 3-dimensional architectures of the iPCs may serve as in vitro models of native SANs. Approach: We will examine four design principles of the native SAN, i) minimum number of iPCs required to pace a given number of neighboring ventricular myocytes, ii) role of non-myocyte population in pacemaking, iii) shape of the SAN, and iv) the need for exit pathways. Our 2D model will consist of patterned monolayers while our 3D model uses patterned cardiac spheroids. Using routine polydimethylsiloxane (PDMS) stenciling techniques, we will create a population of iPCs enclosed by a population of quiescent ventricular cardiomyocytes. The major readouts are i) real-time, whole-cell Ca2+ transients of the entire monolayers, ii) fast, high-resolution optical mapping of the monolayers with a voltage-sensitive dye, and iii) macro-scale, multi-electrode array measurements of field potentials. Our preliminary data indicate that iPC-spheroids are viable for at least three weeks. When a cluster of 15-20 TBX18 spheroids was surrounded by a monolayer of ventricular myocytes, TBX18, but not GFP (control), spheroids were able to pace and drive the neighboring sheet of ventricular myocytes. Successful completion of our project can lead to creation of engineered SA nodes (eSANs) that recapitulate the design principles of the native SAN. In turn, this technology provides a convenient platform on which other SAN design principles may be built toward persistent biological pacemakers.