Reverse-engineering the sinoatrial node with induced pacemaker cells

摘要 背景：哺乳动物的心脏由于几千次自发和自主地跳动 (~10,000)个起搏器细胞。尽管我们对个体心脏如何 起搏器细胞会自动跳动，但对一些起搏器细胞如何 驱动整个心脏的跳动。这个问题，称为“源-汇失配”，是一个根本的问题。 概念一直很难研究，因为研究这些起搏器的吞吐量低得令人痛苦 细胞。这是因为不存在包含心脏起搏细胞的可测试的SAN模型。 和静止的心肌细胞。通常情况下，只有少数天然起搏器细胞可以从 原生SAN和分离的细胞不能培养。最近，我的团队展示了 单细胞培养条件下普通心肌细胞向起搏细胞的转化 Tbx18基因的表达。在这个提案中，我们试图通过利用 新的IPC。我们假设IPC的二维和三维结构可能在体外起作用 原生SAN的模型。 方法：我们将研究本机SAN的四个设计原则：i)所需的最小IPC数量 对给定数量的相邻心室肌细胞进行起搏，II)非心肌细胞群体在 起搏，iii)窦房结的形状，以及iv)出口路径的需要。我们的2D模型将包括 图案化的单分子层，而我们的3D模型使用图案化的心脏球体。使用例程 聚二甲基硅氧烷(PDMS)模版技术，我们将创建一个由 静止期的心肌细胞群。主要读数是i)实时、全细胞的钙离子 整个单层的瞬变，ii)单层的快速、高分辨率光学映射 电压敏感染料，以及iii)场电位的宏观、多电极阵列测量。我们的 初步数据表明，IPC球体至少可以存活三周。当一群15-20的人 TBX18球体被单层的心室肌细胞TBX18包围，而不是GFP(对照)， 球体能够对相邻的心肌细胞进行起搏和驱动。 成功完成我们的项目可以创建工程化SA节点(ESAN)， 概括介绍本征SAN的设计原则。反过来，这项技术提供了一种方便的 可以在其上构建其他SAN设计原则的平台，以实现永久性生物起搏器。

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.