Embryonic Stem Cell Derived Biological Pacemaker for the Heart

胚胎干细胞衍生的心脏生物起搏器

• 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
• 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

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）：可植入的人造起搏器是窦节点功能障碍和传导异常的最新处理方法。然而，它们因手术侵入性修复而引起的有限的神经肿瘤反应性和并发症促使生物起搏器的发展，这些起搏器对神经肿瘤刺激的反应并无限期地整合到心肌中。成人心脏肌肉区域的自发活性的诱导只能纠正问题，因为新形成的起搏器仍然没有保护i）周围心肌细胞的超极化影响，ii）ii）背部传播。因此，我们提出使用具有确定的连接性表达的胚胎干细胞衍生的心肌细胞（ESDC）的聚集体，该表达反映了Sinoatrial节点中建立的细胞间耦合的功能特性。拟议研究的具体目的是：1）确定胚胎干细胞衍生的心肌细胞中的起搏器活性的机制2）确定间隙连接通道的功能特性，该差距可允许ESDC聚集物获得并维持在多细胞心肌细胞制剂中的起搏器优势！ 3）确定促进ESDC和成人心肌细胞杂细胞对之间频率和波形夹带的机制，而无需诱导成人组织中心律不齐的活性！ 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和ESDC和成人心肌细胞的细胞对中进行高分辨率Ca成像；新型基于FRET的荧光IPS传感器研究IPS在起搏器活性中的作用或在成年心肌细胞中心律失常自发活性的过程中；将采用单细胞电流夹具测量来评估ESDC中自发活性的机制，并且将使用双重整个细胞电压夹子测量来表征ESDC之间的细胞间耦合抗性和依赖性依赖性门控为生物性过心者和宿主心脏组织（HL-1 Cell-1 Cells）（HL-1 Cells cells cells cells cells cells cells cells cells cells cell of HIL-1）。但是，多细胞内外起搏器模型的电生理特性将通过HL-1单层和ESDC Pacemaker聚集体的杂细胞培养物的多电极测量来确定。此外，我们将使用稳定转染的ES细胞系，这些ES细胞系在CX43或CX45或过表达CX45的表达不足。