Embryonic Stem Cell Derived Biological Pacemaker for the Heart

胚胎干细胞衍生的心脏生物起搏器

基本信息

批准号：
7516005
负责人：
Kathrin Banach
金额：
$ 34.07万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-08-01 至 2012-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7516005
关键词：
Action Potentials Adult Area Artificial cardiac pacemaker Arts Back Biological Pacemakers Cardiac Cardiac Myocytes Cell Transplants Cell membrane Cells Coculture Techniques Connexin 43 Connexins Coupling Dependence Development Doctor of Philosophy ES Cell Line Endothelin-1 Event Exhibits Fire - disasters Fluorescence Resonance Energy Transfer Frequencies Functional disorder Generations Genes Goals Heart Heart Atrium ITPR1 gene Image In Vitro Invasive Ion Channel Kinetics Knowledge Laser Scanning Confocal Microscopy Measurement Measures Mediating Modeling Muscle Cells Myocardium Nodal Numbers Pacemakers Phenotype Phenylephrine Physiological Preparation Production Property Protein Isoforms Protein Overexpression Replacement Therapy Research Personnel Resistance Resolution Role Ryanodine Receptor Calcium Release Channel Signal Transduction Sinoatrial Node Sinus Stimulus Techniques Testing Tissues Transfection Translating Transplantation Ventricular base embryonic stem cell gap junction channel improved in vitro Model in vivo inhibitor/antagonist monolayer novel prevent programs repaired sensor voltage voltage clamp

项目摘要

DESCRIPTION (provided by applicant): Implantable artificial pacemakers are the state-of the art treatment for sinoatrial node dysfunction and conduction abnormalities. However, their limited neurohumoral responsiveness and complications arising from surgically invasive repair have prompted the development of biological pacemakers that respond to neurohumoral stimuli and indefinitely integrate into the myocardium. The induction of spontaneous activity in an area of the adult cardiac muscle only insufficiently corrects the problem since the newly formed pacemaker remains without protection against i) the hyperpolarizing influences of surrounding cardiomyocytes, and ii) the back propagation of arrhythmic excitation. Therefore we propose the use of aggregates of embryonic stem cell derived cardiomyocytes (ESdCs) with defined connexin expression that reflect the functional properties of the intercellular coupling established in the sinoatrial node. The specific aims of the proposed study are: 1) to determine the mechanism of pacemaker activity in embryonic stem cell derived cardiomyocytes 2) to determine the functional properties of gap junction channels that allow ESdC aggregates to gain and maintain pacemaker dominance in multicellular cardiomyocyte preparations! 3) to determine mechanisms that promote frequency and waveform entrainment between heterocellular pairs of ESdCs and adult cardiac myocytes without induction of arrhythmic activity in the adult tissue! To achieve these aims different experimental techniques will be used. High resolution Ca imaging by laser scanning confocal microscopy in single ESdCs and in cell pairs of ESdCs and adult cardiomyocytes; novel FRET based fluorescent IPS sensors to study the role of IPS in pacemaker activity or during the induction of arrhythmic spontaneous activity in the adult cardiomyocytes; Single cell current clamp measurements will be employed to evaluate the mechanism of spontaneous activity in ESdCs and double whole cell voltage clamp measurements will be used to characterize the intercellular coupling resistance and voltage dependent gating between the ESdCs used as biological pacemakers and the host cardiac tissue (either HL-1 cells or adult cardiomyocytes). The electrophysiological properties of the multicellular in-vitro pacemaker model however will be determined by multielectrode measurements of heterocellular cultures of HL-1 monolayers and ESdC pacemaker aggregates. We will furthermore use stably transfected ES cell lines that are either deficient for the expression of Cx43 or Cx45 or overexpress Cx45.

描述（由申请人提供）：植入式人工起搏器是治疗窦房结功能障碍和传导异常的最先进的方法。然而，它们有限的神经体液反应性和手术侵入性修复引起的并发症促使人们开发出对神经体液刺激做出反应并无限期融入心肌的生物起搏器。在成人心肌区域诱导自发活动仅不足以纠正该问题，因为新形成的起搏器仍然无法防止 i) 周围心肌细胞的超极化影响，以及 ii) 心律失常兴奋的反向传播。因此，我们建议使用胚胎干细胞衍生的心肌细胞（ESdC）的聚集体，其具有明确的连接蛋白表达，反映窦房结中建立的细胞间耦合的功能特性。本研究的具体目标是：1) 确定胚胎干细胞衍生的心肌细胞中起搏器活性的机制 2) 确定间隙连接通道的功能特性，使 ESdC 聚集体在多细胞心肌细胞制剂中获得并维持起搏器优势！ 3) 确定促进 ESdC 异细胞对和成人心肌细胞之间频率和波形夹带的机制，而不诱导成人组织中的心律失常活动！为了实现这些目标，将使用不同的实验技术。通过激光扫描共聚焦显微镜在单个 ESdC 以及 ESdC 和成体心肌细胞的细胞对中进行高分辨率 Ca 成像；基于 FRET 的新型荧光 IPS 传感器，用于研究 IPS 在起搏器活动中或在成人心肌细胞诱导心律失常自发活动过程中的作用；单细胞电流钳测量将用于评估 ESdC 中的自发活动机制，双全细胞电压钳测量将用于表征用作生物起搏器的 ESdC 与宿主心脏组织之间的细胞间耦合电阻和电压依赖性门控。 HL-1 细胞或成体心肌细胞）。然而，多细胞体外起搏器模型的电生理特性将通过对 HL-1 单层和 ESdC 起搏器聚集体的异细胞培养物进行多电极测量来确定。我们还将使用稳定转染的 ES 细胞系，这些细胞系要么缺乏 Cx43 或 Cx45 的表达，要么过度表达 Cx45。