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: 7842168
• 负责人: Kathrin Banach
• 金额: $ 23.49万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-15 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7842168
• 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; Fluorescence Resonance Energy Transfer; Frequencies; Functional disorder; Generations; Genes; Goals; Heart; Heart Atrium; ITPR1 gene; Image; In Vitro; Ion Channel; Kinetics; Knowledge; Laser Scanning Confocal Microscopy; Measurement; Measures; Mediating; Modeling; Muscle Cells; Myocardium; Nodal; Pacemakers; Phenotype; Phenylephrine; Physiological; Preparation; Production; Property; Protein Isoforms; Replacement Therapy; Research Personnel; Resistance; Resolution; Role; Ryanodine Receptor Calcium Release Channel; Ryanodine Receptors; 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; overexpression; 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）：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新技术。然而，他们有限的神经体液反应性和手术创伤性修复引起的并发症，促使生物起搏器的发展，响应神经体液刺激，并无限期地整合到心肌。在成人心肌的区域中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没有针对i）周围心肌细胞的超极化影响和ii）心肌兴奋的反向传播的保护。因此，我们建议使用具有明确连接蛋白表达的胚胎干细胞衍生的心肌细胞（ESDC）的聚集体，其反映了在窦房结中建立的细胞间偶联的功能特性。该研究的具体目的是：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，该通道允许ESdC聚集体在多细胞心肌细胞制备物中获得并维持起搏器优势！3)确定促进ESdCs和成体心肌细胞的异细胞对之间的频率和波形夹带的机制，而不诱导成体组织中的心肌活性！ 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ESdCs和ESdCs与成年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，以研究IPS在起搏器活动中或在诱导成年心肌细胞的自发活动期间的作用;将采用单细胞电流钳测量来评估ESdCs中自发活动的机制，并采用双全细胞电压钳测量来评估ESdCs中自发活动的机制。测量将用于表征用作生物起搏器的ESdc和宿主心脏组织（HL-1细胞或成年心肌细胞）之间的细胞间耦合电阻和电压依赖性门控。然而，多细胞体外起搏器模型的电生理特性将通过HL-1单层和ESdC起搏器聚集体的异细胞培养物的多电极测量来确定。我们还将使用Cx43或Cx45表达缺陷或Cx45过表达的稳定转染的ES细胞系。